Erythropoietin enhances Kupffer cell number and activity in the challenged liver

Erythropoietin (EPO) is the main hormone driving mammalian erythropoiesis, with activity mediated via the surface receptor, EPO-R, on erythroid progenitor cells. Recombinant human EPO is currently used clinically for the treatment of anemia in patients with end-stage renal disease, and in certain cancer patients suffering from anemia induced either by the tumor itself or by chemotherapy. EPO-R expression is also detected in non-erythroid cells, including macrophages present in the peritoneum, spleen, and bone marrow (BM). Here we demonstrate that Kupffer cells (KCs) - the liver-resident macrophages - are EPO targets. We show that, in vitro, EPO initiated intracellular signalling and enhanced phagocytosis in a rat KC line (RKC-2) and in sorted KCs. Moreover, continuous EPO administration in mice, resulted in an increased number of KCs, up-regulation of liver EPO-R expression and elevated production of the monocyte chemoattractant CCL2, with corresponding egress of Ly6C(hi) monocytes from the BM. In a model of acute acetaminophen-induced liver injury, EPO administration increased the recruitment of Ly6C(hi) monocytes and neutrophils to the liver. Taken together, our results reveal a new role for EPO in stimulating KC proliferation and phagocytosis, and in recruiting Ly6C(hi) monocytes in response to liver injury

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

Abstract 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&lt;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&lt;0.05; **depicts p&lt;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. </jats:sec

Resistance of LPS-activated bone marrow derived macrophages to apoptosis mediated by dexamethasone

Glucocorticoids (GC) display pleiotropic effects on the immune system. Macrophages are a major target for GC action. Here we show that dexamethasone (DEX), a synthetic GC, decreased viability of naïve bone marrow-derived macrophages (BMDM), involving an apoptotic mechanism. Administration of DEX together with lipopolysaccharide (LPS) protected BMDM against DEX-mediated cell death, suggesting that activated BMDM respond to DEX differently than naïve BMDM. An insight to the molecular basis of LPS actions was provided by a 7 fold increase in mRNA levels of glucocorticoid receptor beta (GRβ), a GR dominant-negative splice variant which inhibits GRα's transcriptional activity. LPS did not inhibit all DEX-mediated effects on BMDM; DEX significantly reduced the percentage of BMDM expressing high levels of the cell surface markers F4/80 and CD11b and led to a decrease in macrophage inflammatory protein 1 alpha (MIP1-α) mRNA and protein levels. These two DEX-mediated effects were not prevented by LPS. Our finding that LPS did not reduce the DEX-induced elevation of glucocorticoid-induced leucine zipper (GILZ), a mediator of GCs anti-inflammatory actions, may provide an underlying mechanism. These findings enable a better understanding of clinical states, such as sepsis, in which macrophages are activated by endotoxins and treatment by GCs is considered

Erythropoietin Treatment in Multiple Myeloma – Friend or Foe?

Abstract Abstract 5013 Erythropoietin (Epo), mainly produced by the adult kidney, is the major hormone that promotes erythropoiesis. As such, clinical introduction of recombinant human Epo (rHuEpo) and its derivatives (Epoetins), has been a breakthrough in treating patients with anemia, mainly those with chronic kidney failure and cancer patients on chemotherapy. In contrast to 'red flags' suggesting possible detrimental effects of Epo in certain cases of cancer, the hormone was found by numerous studies to have beneficial effects such as neuroprotective and cardio-protective effects. Immune-mediated anti-cancer effects were also documented, as exemplified in multiple myeloma (MM) patients and mouse models. These effects, at least in part, are mediated by Epo receptors (EpoRs) present on cells other than the erythroid lineage. In this respect, we have recently identified functional EpoRs in murine bone marrow-derived macrophages. MM is characterized by clonal proliferation of malignant plasma cells that produce a pathological paraprotein, accompanied by a decrease in the levels of the normal immunoglobulins. Typically this disease is associated with lytic bone lesions. In 5T33 MM, an experimental murine model of MM (producing an IgG2bk paraprotein), mice that were treated with rHuEpo, displayed a 50% decrease in the levels of the pathological K light chain. Notably, the decline in the levels of normal IgA, typically observed in the MM mice, was attenuated in the Epo-treated MM mice. Administration of rHuEpo led to increased levels of liver (three fold increase) and splenic (ten fold increase) macrophages as determined by the number of F4/80/CD11b expressing cells. These findings were associated with an increase in the transcript levels of the Th1 cytokine IFN-g in the bone marrow of Epo-treated 5T33 MM mice as compared to diluent-treated 5T33 MM mice. As MM is typically associated with a Th2 response, Epo may have a beneficial effect in MM, by shifting the balance towards a Th1 response. Myeloma-associated bone disease is considered a major cause of morbidity in MM patients. Using high-resolution computed tomography analysis of recovered femurs we confirmed that the 5T33 MM mice indeed exhibit bone disease. Femur trabecular bone density (bone volume/total volume) of 5T33 MM mice was 43% lower (p=0. 04) than that of their healthy control counterparts (3. 1%±0. 003 versus 4. 4%±0. 008, respectively). This decrease in bone density reflects a 38% increase (p=0. 001) in the total femur volume (4. 38mm3±0. 13 and 2. 72mm3±0. 95 in the 5T33 MM mice, compared to that of healthy mice) and no significant change in trabecular bone volume. Unexpectedly, Epo treatment of the MM mice induced a 34% reduction (p=0. 004) in trabecular bone density compared to the diluent-treated MM mice (2%±0. 007 and 3. 1%±0. 003, respectively) and 41% (p=1E-6) reduction in trabecular number (1. 54mm−1±0. 266 and 2. 62mm−1±0. 23, respectively). This was accompanied by a 19% increase (p=0. 006) in trabecular bone thickness in the Epo-treated MM mice (0. 043mm±0. 005 and 0. 036mm±0. 003, respectively), suggesting a mechanical compensatory mechanism in response to the extensive bone loss. Taken together, our data demonstrate that Epo's impact extends well beyond erythropoiesis. The administration of rHuEpo may be considered as an adjunct treatment to specific anti-MM therapy in cases which require elevation of the erythrocyte counts and an enhancement of the immune system. Yet, a bone protection regimen (bisphosphonates?) should be considered. This study is supported by the Multiple Myeloma Research Foundation, to DN. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Erythropoietin treatment in murine multiple myeloma: immune gain and bone loss

AbstractMultiple myeloma (MM) is a plasma cell malignancy, characterized by osteolytic lesions and monoclonal immunoglobulins. The anemia, accompanying the disease is often treated with recombinant human EPO. Diverse non-erythropoietic effects of EPO have led us to question its combined action on the immune system and bone in the 5T33MM mouse model. EPO administration to MM mice attenuated disease progression as demonstrated by a decrease in serum MM IgG2b, splenic CD138 expressing cells, IL-6 and RORγτ transcripts in bone marrow (BM). IFN-γ transcript levels and macrophages (F4/80+CD11b+) in the BM both increased ~1.5 fold in the EPO-treated MM mice. In-vitro, EPO stimulated phagocytosis of 5T33MM cells (+30%) by BM-derived macrophages. In contrast, high-resolution microCT analysis of distal femurs revealed EPO-associated bone loss in both healthy and 5T33MM mice. EPO significantly increased expression of the osteoclastogenic nuclear factor-kappa B ligand (RANKL) in healthy mice, but not in MM mice, likely due to antagonizing effects on MM progression. Thus, in MM, EPO may act as a double-edged-sword stimulating immune response, while accelerating bone resorption, possibly via direct action on BM macrophages. This study supports a prudent approach of treating anemia in MM patients, aiming to maintain EPO-associated anti-MM effects, while considering bone damage.</jats:p

Immune Dysfunction in Patients with MDS Is Partially Corrected in EPO-Treated Patients: Differences According to WHO Classification

Abstract Background: The immune system has been shown to be involved in the pathogenesis of myelodysplastic syndromes (MDS), and is also affected by the disease. Recombinant erythropoietin (rHuEPO), or in general, erythroid stimulating agents (ESAs) have become a standard treatment for anemic patients with MDS. They were found to improve anemia, quality of life, and possibly survival. We have previously demonstrated that EPO has effects on cellular and humoral immunity and specifically, on immune function in patients with multiple myeloma (MM). Here we report our findings demonstrating the effect of ESAs on T cell (CD4+, CD8+ and CD4+CD25+) number and function in patients with MDS. Patients and Methods: We examined three groups: healthy subjects ('Control', 20 participants), MDS patients without ESA treatment ('MDS', 13), and MDS patients treated with an ESA ('MDS+EPO', 17). All diagnosed patients gave informed consent as approved by our IRB. Cell numbers were evaluated with flow cytometry. In a subset of patients, cell activation was assessed in response to phytohemagglutinin (PHA) by examining CD69 expression in both CD4+ and CD8+ cells. The co-stimulatory marker, CD28, and the inhibitory marker CTLA-4 (CD152) were evaluated as well. We also examined World Health Organization (WHO) subgroups, refractory anemia (RA) and RA with ringed sideroblasts (RARS) versus more advanced disease. Results: CD4+ and CD8+ T cell levels are reduced and increased respectively in MDS patients compared to control, and these changes are reversed in MDS+EPO (Table 1, CD4+, p&lt;0.01; CD8+, p=0.05). The CD4+:CD8+ ratio (Table 1) is reduced and nearly equalized in MDS (1.16), but approaches that of the control (2.24) in MDS+EPO (1.94). CD4+CD25+ T cell numbers (including regulatory T cells), were lower in MDS patients and improve in the MDS+EPO group (Table 1). In vitro activation of T cells (CD4+CD69+ and CD8+CD69+) achieves an approximately 15-fold increase in healthy subjects. MDS patients without EPO sustained only a 7.17 fold increase in CD4+ activation versus 13.64 fold for the MDS+EPO group (p&lt;0.01); for CD8+ T cells, 10.20 fold (MDS) versus 18.56 fold (MDS+EPO, p&lt;0.01). The expression of the co-stimulatory marker CD28 was decreased in both CD4+ and CD8+ T cells in MDS, and approached normal in MDS+EPO in CD4+ T cells (Table 1). There was no significant change in inhibitory CTLA-4 (CD152) expression among the groups (not shown). Subgroup analysis demonstrated that ESA has a similar effect on CD4+ and CD8+ cells and their ratio in both RA/RARS and more advanced disease, similar to those of the whole cohort (Table 2, green). On the other hand, some parameters were affected by ESA only in one subgroup (Table 2, blue): The ESA effect on CD4+CD25+ cells was evident only in patients with advanced disease (Table 2, blue). ESA affected CD4+ and CD8+ cell stimulation (CD69) in RA/RARS, similar to that seen in the whole cohort (Table 2, blue). Of note, in more advanced disease, CD4+ and CD8+ cells achieved stimulation in the MDS group not treated with ESA, with no difference between MDS and MDS+EPO. This finding needs to be further addressed in larger cohorts and with additional markers of activation. Conclusions: MDS patients display T-cell abnormalities that are improved upon EPO treatment. MDS is a heterogeneous disease where the immune system both affects and is affected by the disease. As such, treatment with ESAs might ameliorate not only the anemia, but also the immune deficiencies and perhaps the disease itself. Future studies will clarify the immunomodulatory role of ESA in the various stages of MDS. Disclosures No relevant conflicts of interest to declare. </jats:sec

Nanoscale CAR Organization at the Immune Synapse Correlates with CAR-T Effector Functions

T cells expressing chimeric antigen receptors (CARs) are at the forefront of clinical treatment of cancers. Still, the nanoscale organization of CARs at the interface of CAR-Ts with target cells, which is essential for TCR-mediated T cell activation, remains poorly understood. Here, we studied the nanoscale organization of CARs targeting CD138 proteoglycans in such fixed and live interfaces, generated optimally for single-molecule localization microscopy. CARs showed significant self-association in nanoclusters that was enhanced in interfaces with on-target cells (SKOV-3, CAG, FaDu) relative to negative cells (OVCAR-3). CARs also segregated more efficiently from the abundant membrane phosphatase CD45 in CAR-T cells forming such interfaces. CAR clustering and segregation from CD45 correlated with the effector functions of Ca++ influx and target cell killing. Our results shed new light on the nanoscale organization of CARs on the surfaces of CAR-Ts engaging on- and off-target cells, and its potential significance for CAR-Ts’ efficacy and safety

Erythropoietin Mediated Increase in Monocyte-Derived Liver Macrophages; A Role for Kupffer Cells?

Abstract Erythropoietin (EPO) is the major hormone that drives mammalian erythropoiesis, via its surface receptor, EPO-R. It is mainly used for treating anemia associated with chronic renal failure and certain malignancies, although this latter indication is currently disputed. EPO-Rs were also found in non-erythroid cells, including dendritic cells and bone marrow macrophages (Lifshitz, 2008; 2010). Here we addressed the effect of EPO on hepatic-macrophages, namely resident liver macrophages (Kupffer cells) and liver monocyte-derived macrophages (MFs). Utilizing the rat Kupffer cell line (RKC-2) we demonstrate that these cells express EPO-R transcripts and cell surface EPO-R, as detected by our novel EPO-R antibody (GM1012; Maxwell 2015). EPO treatment of the RKC-2 cells led to a 1.5±0.06 fold increase (p&lt;0.05) in EPO-R mRNA levels and a 2±0.01 fold decrease (p&lt;0.01) in surface EPO-R levels. Stimulation of the cells with EPO induced a 15%±0.06 (p=0.01) increase in transcript levels of CCL-2 (a chemo attractant for monocytes) and a 15%±0.6 (p&lt;0.05) increase in the levels of the secreted chemokine. EPO treatment also enhanced cellular activity of the RKC-2 cells as manifested in a 50%±0.13 (p&lt;0.01) increase in cell migration, an increase in phagocytosis of microbeads (40%±0.08, p&lt;0.01) and of E.coli (13%±0.05, p&lt;0.01). Finally, in vivo experiments in which C57BL/6 mice were injected 3 times a week with 180U recombinant Human EPO (rHuEPO), demonstrated an EPO-induced selective increase in MFs (19.5%±0.01, p=0.05), but not in Kupffer cells. Elevated CCL-2 in sera of EPO-injected mice (2.1 fold increase, p&lt;0.01) supports a mechanism by which EPO stimulates Kupffer cells to increase secretion of CCL-2, which in turn enhances recruitment of monocytes to the liver and their subsequent differentiation into MFs. The present study points to a new as hitherto unexplored action of EPO on two separate liver macrophage populations (Kupffer cells and MFs) which play crucial and distinct roles in liver homeostasis and immunity as well as in liver pathologies. EPO selective actions on liver macrophages thus call for future studies on the effects of this hormone on inflammation in the liver with a therapeutic goal to enhance liver immunity. Disclosures Mittelman: Celgene: Research Funding, Speakers Bureau; GlaxoSmithKline: Research Funding; Johnson &amp; Johnson: Research Funding, Speakers Bureau; Novartis Pharmaceuticals Corporation: Research Funding; Roche: Research Funding; Amgen: Research Funding. </jats:sec

Erythropoietin directly stimulates osteoclast precursors and induces bone loss

Erythropoietin (EPO) primarily regulates red blood cell formation, and EPO serum levels are increased on hypoxic stress (e.g., anemia and altitude). In addition to anemia, recent discoveries suggest new therapeutic indications for EPO, unrelated to erythropoiesis. We investigated the skeletal role of EPO using several models of overexpression (Tg6 mice) and EPO administration (intermittent/continuous, high/low doses) in adult C57Bl6 female mice. Using microcomputed tomography, histology, and serum markers, we found that EPO induced a 32%-61% trabecular bone loss caused by increased bone resorption (+60%-88% osteoclast number) and reduced bone formation rate (-19 to -74%; P < 0.05 throughout). EPO targeted the monocytic lineage by increasing the number of bone monocytes/macrophages, preosteoclasts, and mature osteoclasts. In contrast to the attenuated bone formation in vivo, EPO treatment in vitro did not inhibit osteoblast differentiation and activity, suggesting an indirect effect of EPO on osteoblasts. However, EPO had a direct effect on preosteoclasts by stimulating osteoclastogenesis in isolated cultures (+60%) via the Jak2 and PI3K pathways. In summary, our findings demonstrate that EPO negatively regulates bone mass and thus bears significant clinical implications for the potential management of patients with endogenously or therapeutically elevated EPO levels.-Hiram-Bab, S., Liron, T., Deshet-Unger, N., Mittelman, M., Gassmann, M., Rauner, M., Franke, K., Wielockx, B., Neumann, D., Gabet, Y. Erythropoietin directly stimulates osteoclast precursors and induces bone loss