ALBERT

Description: Inflammatory states seen in infection and other chronic disorders are often characterized by a condition called anemia of inflammation (AI). The iron deficiency in AI is predominantly due to an altered balance of the cytokine-interleukin-6 (IL6) and the hormone hepcidin (Hamp). IL6 has been implicated in inducing expression of hepcidin, which degrades the iron exporter ferroportin. We have previously shown that lack of IL6 or hepcidin in knockout mouse models (IL6-KO and Hamp-KO) injected with the heat-killed pathogen Brucella abortus (BA) results in improved recovery from anemia. This recovery was different in IL6-KO and Hamp-KO mice, suggesting that the two proteins contribute independently to AI. Here, we formally validated the independent role of IL6 and Hamp in AI by generating a double-knockout (DKO) mouse model lacking the expression of both. The DKO mice showed the most ameliorated phenotype following BA administration. BA-treated-DKO mice showed an increased number of erythroblasts in the bone marrow (BM) and spleen as seen by flow cytometry, in comparison to IL6-KO and Hamp-KO. Concurrently, compared to WT, Hamp-KO and IL6-KO animals, in DKO mice the reticulocyte count was already increased by week-2. The anemia induced by the pathogen by week-1 was less severe in DKO mice. Moreover, both hemoglobin and RBC values measured at week-2 were the highest in DKO, followed by Hamp-KO and then IL6-KO. We also investigated RBC lifespan in these animals by measuring the turnover of biotinylated RBC over time. The turnover of the biotinylated RBC occurred in two phases. In the initial phase the percentage of biotinylated and non-biotinylated RBCs in the BA-treated animals remained the same, while in the second phase it decreased, indicating production of new RBCs. In Hamp-KO and the IL6-KO mice the first phase lasted for 7 days and 4 days respectively, while in DKO animals the percentage of biotinylated RBC had already started to decrease by day 4, indicating an accelerated production of new RBCs compared to single KO mice. Additionally, we used the RodentMAP®-v.3.1 (MyriadRBM) to quantify 51 serum inflammatory biomarkers, and Ingenuity Pathway Analysis to identify pathways activated in single KO as well as DKO mice. Of the top 10 pathways activated in all three models, 4 present in the IL6-KO were also activated in the DKO model, and the remaining 6 were unique. Five of the pathways that came up in Hamp-KO were also activated in DKO mice and the remaining 5 were unique. For instance, both DKO and Hamp-KO mice showed activation of Hmgb1 signaling, suggesting a response to limit inflammation and reduce tissue damage. Moreover, both the DKO and IL6-KO models showed activation of granulocyte adhesion and diapedesis, the former suggesting an inflammatory response associated with the infection while the latter possibly indicating mobilization of cells in response to the infection. A unique pathway activated in DKO mice was that associated with increased production of pluripotent stem cells, likely triggered by the damage observed to the BM and anemia and potentially responsible for the accelerated recovery observed in these animals. In conclusion, these results suggest that the absence of both IL6 and Hamp not only is associated with activation of pathways in common with the single KO, but also with unique features triggered by the concurrent depletion of the two genes. Potential clinical implications will be discussed. Disclosures Rivella: Medgenics Pharmaceuticals: Consultancy; Novartis Pharmaceuticals: Consultancy; Merganser Biotech: Other: Stock options; isis Pharmaceuticals: Consultancy; Bayer Healthcare: Consultancy, Research Funding.

Description: After blood loss, the production of red cells must be increased by stress erythropoiesis. This phenomenon is associated with increased proliferation and reduced differentiation of the erythroblasts, leading to a net increase in the number of progenitor erythroid cells and red cells (erythron). In normal conditions, after expansion of the pool of erythroblasts, these cells eventually differentiate to erythrocytes and the anemia resolves. However, in diseases such as β−thalassemia, production of healthy mature erythrocytes is impaired, resulting in anemia. Over time, the expansion, rather than the differentiation, of the erythron further exacerbates the ineffective erythropoiesis (IE), reducing the ability of the erythroid progenitors to generate erythrocytes. Interrupting the interaction between macrophages and erythroblasts (MEI) in thalassemia models is efficacious in reducing IE and alleviating the disease phenotype. We speculate that these molecules are also responsible for the homing of erythroid progenitor cells to extramedullary organs, such as the spleen and liver. Our studies in erythroblasts indicate that integrin beta−1 (Itgβ1) and also intracellular molecules such as focal adhesion kinase (Fak1), Talin−1 and Sharpin might play a role in stress erythropoiesis. Furthermore, there is increased interaction between Itgb1 and Fak1 in erythroblasts co−cultured with macrophages as demonstrated by immunocytochemistry and in vitro proximity ligation assays. In addition, targeting either Itgβ1 or Fak1 prevents expansion of erythroid cells when cultured in the presence of macrophages. Strikingly, using Itgβ1 together with Ter119 as selection parameters in flow cytometry, a distinct subset of erythroblasts, not discernable using CD44 or CD71, was observable, which we found to be part of the mixed orthochromatic erythroblast/reticulocyte population as determined with CD44 expression. Enucleation of erythroblasts was accompanied by a marked loss of Itgβ1 expression, indicating that Itgβ1 may be involved in erythroblast enucleation and differentiation. We crossed Hbbth3/+ mice with animals in which Itgβ1 or Fak1 were floxed and carrying an inducible Cre−recombinase (Mx1−Cre). From these animals, we investigated three different models; two obtained from breeding (Hbbth3/+−Itgβ1fl/fl−Mx1−Cre and Hbbth3/+−Fak1fl/fl−Mx1−Cre) and one by bone marrow transplant (BMT) of hematopoietic stem cells (HSCs) of Hbbth3/+−Itgβ1fl/fl −Mx1−Cre animals into wt mice to generate thalassemic animals that expressed the floxed Itgβ1 only in hematopoietic cells. After serial administration of Poly(I)−Poly(C) [poly(I:C)] the animals were analyzed for their erythropoiesis in the bone marrow and spleen. All the animals treated with poly(I:C) showed populations of Itgβ1 or Fak1 negative cells in the bone marrow and spleen. This indicated that all the HSCs were successfully depleted of the Itgβ1 or Fak1 gene. Interestingly, the spleen weight of all the treated animals was reduced, on average, 50% compared to untreated thalassemic mice. Similar results were seen also in Hbbth3/+−Itgβ1fl/fl−Mx1−Cre animals generated through BMT. Therefore, Itgβ1 and Fak1 might contribute to the pathophysiology of thalassemia and their removal might result in reduced stress erythropoiesis, erythroid proliferation and, as a consequence, amelioration of splenomegaly. Iron analysis and quantification of Erythroferrone (ERFE) are in progress to evaluate the impact of depleting Itgβ1 and Fak1 on these mechanisms. We are now in the process of identifying compounds that target MEI and, in particular, Itgβ1. Such molecules might be utilized for development of new treatments for thalassemia or additional disorders of aberrant erythropoiesis. Disclosures Feldman: Bayer ealthCare Phamaceuticals Inc.: Employment. Rivella:isis Pharmaceuticals: Consultancy; Merganser Biotech: Other: Stock options; Novartis Pharmaceuticals: Consultancy; Medgenics Pharmaceuticals: Consultancy; Bayer Healthcare: Consultancy, Research Funding.

Description: Macrophages have been implicated in erythropoiesis historically as a mediator of iron recycling and a key component of the erythroblastic island-consisting of a central macrophage surrounded by erythroid cells in different stages of differentiation. Recently we and others have shown that macrophages contribute to stress erythropoiesis, and such contributions extend beyond the known macrophage function of iron recycling. This finding necessitates the investigation of processes within a macrophage itself that might facilitate stress erythropoiesis and the characterization of macrophage transcriptome signatures associated with the same. Macrophages in the bone marrow have been identified previously as CD115LowGR1LowF4/80High cells. Extending this to the spleen, we sorted and sequenced singlet macrophages in the bone marrow and spleen of two mice models of stress erythropoiesis: acute stress induced by retro orbital phlebotomy and chronic stress by bone marrow transplanted Polycythemia Vera. We found that splenic macrophages show more significant changes upon induction of stress (both acute and chronic) in comparison of macrophages in the bone marrow. The mean of normalized count of each gene when plotted against fold change (Figure 1) show that the acute and chronically stressed splenic macrophages differ significantly over non stressed macrophages but not over each other. Fig 1: Acute over non stressed Chronic over non stressed Acute over non stressed Fig 1:. Acute over non stressed Chronic over non stressed Acute over non stressed We further found that stressed splenic macrophages have a characteristic gene expression profile associated with erythropoietic stress, common to both models of induced and chronic stress. There were a total of 2602 genes showing significant changes in expression upon induction of acute stress and a total of 2196 genes showing significant changes in expression upon chronic stress. Out of these, 1529 genes common to both acute and chronically stressed splenic macrophages showed an expression profile common to both acute and induced stress. (Fig 2) Fig 2. Fig 2. We used the open bioinformatics resource DAVID and Ingenuity Pathway Analysis (IPA) to look at biological processes encompassing our significantly up regulated candidates (FDR 10%), upstream analysis of our candidates and further network analysis. Some biological processes of highest significance were cellular growth and proliferation, hematological system development and function, inflammatory response, response to infection, cellular adhesion, cellular movement and immune cell trafficking. We validated some of our key candidates under these biological processes by flow cytometry, such as the iron exporter ferroportin, the receptor for the anti inflammatory cytokine, IL-10 and the cellular adhesion molecule ICAM-1. Some transcription factors significantly upregulated were Hes1, Nfkb1A, RelB, Bcl3 to mention a few. Upstream analysis using IPA predicted activation of transcription factors such as Notch1, Jun, RelA etc. Along with further validation of our key candidates, we are currently investigating macrophage gene expression in another model of chronic stress, beta thalassemia and how this profile associated with the physiological condition of erythropoietic stress compares to those under in vivo models of classical and alternative macrophage activation. Disclosures Casu: Merganser Biotech: Research Funding; Isis Pharmaceuticals Inc.: Research Funding. Rivella:Merganser Biotech: Consultancy, Research Funding, Stock options, Stock options Other; Bayer: Consultancy, Research Funding; Isis Pharmaceuticals. Inc.: Consultancy, Research Funding.

Description: Macrophages, strategically positioned in the center of erythroblastic islands while surrounded by developing erythroblasts (EB), are important for both steady state and stress erythropoiesis. In addition to their function in iron recycling and disposal of nuclei expelled by the maturing red cells during differentiation, macrophages are able to drive erythropoietic activity directly, making them, along with erythropoietin and iron, key regulators of erythropoiesis. This stress erythropoiesis-supporting macrophage activity (SEMA) has been demonstrated recently; there is, however, only limited understanding with regard to the exact cellular mechanism by which the macrophage activity is conveyed to the proliferating erythroid cells. Erythroblastic signaling through integrin β1 (Itgb1) and, further downstream, focal adhesion kinase-1 (Fak1), has previously been linked to stress erythropoiesis. Therefore, the current study explored the role of the Itgb1/Fak1 pathway in the macrophage-EB interplay, as well as its therapeutic potential in disorders marked by chronic stress erythropoiesis (CSE). Mice with beta-thalassemia intermedia (Hbbth3/+ or BTI), which present with ineffective erythropoiesis, characterized by high proliferation but limited differentiation of (CD71+) erythroblasts, anemia, and splenomegaly, were used as a model of CSE. In BTI mice, the depletion of macrophages by intravenous administration of clodronate-loaded liposomes (Clod) resulted in an augmentation of thalassemic erythroid maturation, characteristically marked by a decrease of immature (CD71+Ter119+) and increase of mature (CD71-Ter119+) erythroid cells in bone marrow; a reduction in actively cycling erythroblasts in the spleen, i.e. percentage of cells in S-phase (Clod (20%) compared to PBS (40%) PBS, p

Description: After blood loss, the production of red cells must be increased by stress erythropoiesis. This phenomenon is associated with increased proliferation and reduced differentiation of the erythroblasts, leading to a net increase in the number of progenitor erythroid cells and red cells (erythron). In normal conditions, after expansion of the pool of erythroblasts, these cells eventually differentiate to erythrocytes and the anemia resolves. However, in diseases such as β-thalassemia, production of healthy mature erythrocytes is impaired, resulting in anemia. Over time, the expansion, rather than the differentiation, of the erythron further exacerbates the ineffective erythropoiesis (IE), reducing the ability of the erythroid progenitors to generate erythrocytes. Interupting the interaction between macrophages and erythroblasts (macrophage-erythroblast interaction, MEI) in thalassemia models is efficacious in reducing IE and alleviating the disease phenotype. Targeting MEI, using a number of approaches, caused a significant improvement in blood parameters in β-thalassemia intermedia (BTI) mouse models (Hbbth3/+) and a rapid and dramatic improvement in splenomegaly, an outcome that is relevant for clinical practice. Importantly, MEI is not critical for hematopoiesis under non-stress conditions, and ablation of this interaction in normal mice showed minimal effects on blood parameters. As our initial observations indicate that MEI is essential to support stress erythropoiesis, we investigated adhesion molecules that might activate downstream pathways in erythroblasts that regulate cell proliferation. We also speculate that these molecules are also responsible for the homing of erythroid progenitor cells to extramedullary organs, such as the spleen and liver. Our studies in erythroblasts indicate that integrin beta 1 (Itgb1) and also intracellular molecules such as Fak1, Talin1 and Sharpin might play a role in stress erythropoiesis. There is increased interaction between Itgb1 and Fak1 in erythroblasts co-cultured with macrophages as demonstrated by immunocytochemistry and in vitro proximity ligation assays. In addition, targeting either Itgb1 and Fak1 prevents expansion of erythroid cells when cultured in the presence of macrophages. Strikingly, using Itgb1 together with Ter119 as selection parameters in flow cytometry, a distinct subset of erythroblasts, not discernable using CD44 or CD71, was observable, which we found to be part of the mixed orthochromatic erythroblast/reticulocyte population as determined with CD44 expression. More specifically, when measuring the content of DNA, we were able to demonstrate that enucleation of erythroblasts was accompanied by a marked loss of Itgb1 expression, indicating that there may be an important role for Itgb1 in erythroblast enucleation, and differentiation in general. Lack of Itgb1 in thalassemic mice prevents erythroid cells from homing to and expanding in the spleen, the major source of chronic stress erythopoiesis in this disorder. In particular, such a role of Itgb1 is supported by our analysis of thalassemic mice in which this molecule was partially depleted by induction of the Cre recombinase. These animals were generated by crossing th3/+ mice with animals in which Itgb1 was floxed and carrying an inducible Cre-recombinase (Mx1-CRE). We utilized the BM of these animals (Hbbth3/+, Itgb1fl/fl, Mx1-CRE) to generate thalassemic animals that expressed the floxed Itgb1 only in hematopietic cells. After serial administration of polyI:C the animals were analyzed for their erythropoiesis in the bone marrow and spleen. Interestingly, all the animals analyzed show chimeric populations of Itgb1 positive and negative erythroid cells in the bone marrow. This indicated that not all the HSCs were successfully depleted of the Itgb1 gene. However, when we investigated Itgb1 in the spleen, we observed only erythroid cells positive for the expression of this adhesion molecule. This last observation strongly suggests that depletion of Itgb1 prevents homing and expansion of erythroid cells in the spleen and drugs that by inhibit Itgb1 could reduce erythroid spleen colonization, splenomegaly and limit erythropoiesis. We are now in the process of identifying compounds that target MEI. Such molecules might be utilized for development of new treatments for thalassemia or additional disorders of aberrant erythropoiesis. Disclosures Casu: Merganser Biotech : Research Funding; Isis Pharmaceuticals, Inc.: Research Funding.

Description: Both β-thalassemia intermedia and major are characterized by formation of hemichromes in erythroid cells, impairing their survival and the lifespan of red blood cells (RBC). Minihepcidins (MH) are novel compounds that function as hepcidin agonists and reduce iron absorption and transferrin saturation. Hbbth3/+ mice show features of β-thalassemia intermedia, such as ineffective erythropoiesis (IE), anemia and reduced hepcidin synthesis, but do not require blood transfusion for survival (non-transfusion dependent thalassemia or NTDT). As we have previously shown, the administration of MH in these animals decreased transferrin saturation, erythroid iron intake, heme synthesis and hemichrome formation, with a significant beneficial effect on RBC quality, lifespan and anemia (Casu et al, Blood 2016). In order to test if this approach could also benefit animals affected by β-thalassemia major we focused on generating a model that exhibited a low production of RBCs, severe anemia and a blood transfusion requirement for survival, as in patients affected by transfusion dependent thalassemia or TDT. We have previously shown that engraftment of Hbbth3/th3fetal liver cells (FLCs) into normal mice leads to a very severe anemia that requires blood transfusion for survival (Gardenghi et al, Blood 2007). However, Hbbth3/th3FLCs do not contain any adult or fetal-globin genes and are unable to make hemoglobin in the transplanted animals, in contrast to human β-thalassemia. Therefore, animals cannot benefit from therapies that decrease hemichrome formation and target IE such as MH. To overcome this limitation, we crossed Hbbth3/+mice with additional models of NTDT, indicated as Hbbth1/th1and Hbbth2/+. These mice harbor alternative mutations so that the synthesis of the mouse b-globin genes is different in each model. Hbbth1/th2and Hbbth1/th3pups were alive at birth, but unable to survive more than a couple of days even with the support of blood transfusion. However, recipient transgenic animals expressing GFP and engrafted with Hbbth1/th2andHbbth1/th3FLCs showed the desired phenotype 3 months post-transplant including production of GFP- RBCs (with less than 2% of host GFP+ RBC) and a different degree of anemia, respectively 5.6±0.5 g/dL and 3.1±1.5 g/dL. In the long term these animals require blood transfusion for survival. Therefore these models are useful to test drugs that have the potential to modify erythropoiesis and RBC production. Ten weeks following engraftment with Hbbth1/th2FLCs, mice were treated for six weeks with two different doses of MH (5.25 mg/kg and 2.625 mg/kg administered every other day) in absence of blood transfusion. Animals treated with vehicleshowed severe ineffective erythropoiesis and worsening anemia over 6 weeks (from 5.6±0.5 g/dL on D0 to 5.0±0.7 g/dL on D42 of treatment). In contrast, animals treated with MH showed reversal of anemia at 3 weeks (6.6±0.3 g/dL and 6.1±0.6 g/dL in the 5.25 mg/kg and 2.625 mg/kg group, respectively, compared to 5.3±0.9 g/dL in controls), while at 6 weeks the differences were reduced compared to vehicle treated mice (6.0±0.4 g/dL and 5.7±0.5 g/dL in the 5.25 mg/kg and 2.625 mg/kg group, respectively, compared to 4.9±0.7 g/dL in controls). The RBC number followed the same trend. Furthermore, the RBC morphology of animals treated with MH was improved compared to control animals. At 6 weeks, splenomegaly was also improved in the treatment groups (13.8±2.7 mg and 16.9±2.7 mg respectively in the 5.25 mg/Kg and 2.625 mg/Kg group compared to 26.9±3.5 mg in controls). Comparing the data at 3 versus 6 weeks, we speculate that, while the MH has a positive effect on RBC quality and production, this is insufficient, in the long term, to prevent the severe splenomegaly and the consequent entrapment of the RBC, which exacerbates the anemia over time. However, we hypothesized that administration of MH could have longer lasting beneficial effects in presence of blood transfusion, which would limit the splenomegaly. Presently, we are testing this hypothesis using both the Hbbth1/th2and Hbbth1/th3models. Complete characterization of these models and their parameters (CBC, erythropoiesis, iron metabolism and organ iron content) is in progress. In conclusion, these models can be utilized to characterize severe thalassemia phenotypes and new drugs that have the potential to ameliorate IE and improve RBCs generation. Disclosures MacDonald: Merganser: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: The current treatment of β-thalassemia only partially mitigates the phenotype of the disease, making the need for novel therapeutic agents imperative. The investigational drug Luspatercept (ACE-536) is a ligand trap that contains the modified extracellular domain of activin receptor IIB (ACVR2B) and induces red blood cell production in an erythropoietin independent fashion. ACE-536 binds with high affinity to members of the transforming growth factor (TGF) β superfamily and therefore alters activin/GDF signaling through the intracellular SMAD complex. In search of the specific ligands, recent studies in a mouse model of β thalassemia intermedia identified growth differentiation factor 11 (GDF11) as a possible target of the drug. It has been proposed that GDF11 is overexpressed in thalassemic erythroblasts and inhibits terminal erythroid maturation via SMAD complex phosphorylation. A negative role of GDF11 in erythropoiesis has been postulated also in myelodysplastic syndrome (MDS). We recapitulate, by a genetic approach, the phenotype of thalassemic and MDS mice treated with RAP-536, the murine counterpart of ACE-536. We generated and analyzed animals with GDF11 deletion in erythroid cells (Hbbth3/+ Gdf11fl/flEpoR-Cre and NUP98-HOXD13 Gdf11fl/flEpoR-Cre) and in all hematopoietic tissues (Hbbth3/+Gdf11fl/flVav-Cre and NUP98-HOXD13 Gdf11fl/flVav-Cre). We did not detect any changes in red blood cell number, reticulocyte count, hemoglobin or hematocrit levels compared to thalassemic or MDS mice in absence of the floxed gene. Focusing on thalassemic mice, administration of RAP-536 significantly improved the anemia and other hematopoietic parameters in the peripheral blood, decreased spleen size and ameliorated ineffective erythropoiesis as indicated by an increased ratio of mature to immature splenic erythroblasts analyzed by flow cytometry. Similar endpoints were seen comparing floxed and non-floxed animals treated with RAP-536. Therefore, the lack of GDF11 in erythroid or bone marrow derived cells did not prevent a response to the drug. To assess the effect of a pancellular absence of GDF11, we are currently in process of generating a model of β-thalassemia with total Gdf11 deletion. To investigate the possible effects of RAP-536, we treated erythroid cells derived from normal or thalassemic patients with the drug. Erythroid cell viability, number, differentiation and cell cycle remained unvaried. Of note, we did not detect significant expression of GDF11/Gdf11 in human and mouse erythroid cells. To investigate the effect of an exogenous source of GDF11 production on erythroid cells, we treated murine erythroleukemia (MEL) cells with recombinant GDF11. Upon treatment we observed phosphorylation of the SMAD2/3 complex by western blot. This effect was hindered by co-treatment of GDF11 with RAP-536. We further assessed the effects of RAP-536 on the mouse erythroid transcriptome, using RNA seq analysis in splenic erythroid populations. After administration of a single dose of RAP-536, thalassemic mice were euthanized. We used flow cytometry to identify possible alterations on differentiating erythroid populations in the spleen. Notably, between 60 and 72h we observed reduced numbers of basophilic and increased numbers of polychromatophilic erythroblasts. Analysis at 60h revealed that signal transducer and activator of transcription 5a (Stat5a), cyclin-dependent kinase 6 (Cdk6) and other cell cycle-related and metabolic genes were increased in the basophilic erythroid progenitors treated with RAP-536. This effect suggests that RAP-536 promotes proliferation and/or differentiation of erythroblasts. Thus, our genetic analyses suggest that lack of GDF11 may be required but not sufficient to improve erythropoiesis. Furthermore, erythroid cells do not produce but can respond to exogenous GDF11, likely synthesized by non-erythroid cells and under conditions of ineffective erythropoiesis. Even though we detected in vitro effects, these may not mimic physiological effects, as experimental conditions may not correlate with GDF11 concentrations in vivo. As additional ligands have been proposed (such as GDF8 and Activin B), our future studies will focus on the potential role of these molecules. Altogether, these results reveal a potential alternative target of action for ACE-536 and may lead to the discovery of new therapeutic molecules. Disclosures Suragani: Acceleron Pharma: Employment, Equity Ownership. Kumar:Acceleron Pharma: Employment, Equity Ownership.

Description: Anemia of inflammation, also known as anemia of chronic disease is the second most common anemia after iron deficiency anemia. The predominant regulators of AI are the cytokine-interleukin-6 (IL6) and the hormone hepcidin (Hamp). IL6 has been implicated in inducing expression of hepcidin. Published data from our lab have shown that lack of IL6 or hepcidin in knockout mouse models (IL6-KO and Hamp-KO) injected with the heat-killed pathogen Brucella abortus(BA) results in recovery from anemia but interestingly the pattern of the recovery was different in IL6-KO and Hamp-KO mice, suggesting that the two proteins contribute independently to AI. Here, we validated the independent role of IL6 and Hamp in AI by generating a double-knockout (DKO) mouse model lacking the expression of both. In the first few days following BA administration, we observed severe reduction in the total number of BM cells in each model followed by a slow recovery in erythroid and multilineage hematopoietic cells. The recovery, initially, was more sustained in the BA-treated-DKO model. In particular, in the first week, BA-treated-DKO mice showed an increased number of erythroblasts in the bone marrow (BM) and spleen as seen in comparison to IL6-KO and Hamp-KO. IL6-KO mice showed an intermediate recovery profile when compared to DKO and Hamp-KO, the last one showing the worst profile in the BM. Interestingly, when the reticulocyte count in the DKO mice was compared to that of IL6-KO and Hamp-KO mice, it showed a biphasic trend, with a significant increase in number during the 2nd week, followed by a significant reduction during the 3rd week. We hypothesized that the initial surge in reticulocyte count in DKO was due to lack of hepcidin, which increases iron availability to erythroid cells, and concurrent lack of IL6, which favors BM erythropoiesis in presence of inflammatory stimuli. However, we also speculated that the excess of iron (as NTBI), which accumulates during the first two weeks, leads to oxidative stress and erythroid cell death in presence of inflammatory cytokines, despite the absence of IL6. We also surmised that, during the second week, a second wave of inflammatory cytokines is triggered by the adaptive response in response to the BA that would explain the negative effect on erythropoiesis after the initial recovery. To assess this hypothesis, we utilized an inflammation panel to analyze the cytokine expression in WT animals treated with PBS or BA at 6 hours, 24 hours and then around ~2 weeks. The cytokine levels were normalized after 24 hours. However, around two weeks, we observed a novel surge of cytokines such as IFN-g and TNFa in the BA treated mice, indicating their role in innate (immediate effect; 6 hours) and adaptive immune response, which activated a second wave of inflammation (around 2 weeks, during the recovery of hematopoiesis in the BM). Interestingly, while we observed oxidative stress and defective erythropoiesis in the bone marrow, this was not seen in the spleen, where increased and extramedullary erythropoiesis sustained some level of RBC production. Since the BA-treated-IL6-KO did not show any major defect in the BM after two weeks, we challenged them with administration of iron dextran. Upon treatment, also the IL6-KO mice treated with both BA and iron dextran shown increased production of reactive oxygen species as well as a defect in bone marrow erythropoiesis, similarly as in DKO or Hamp-KO mice, thereby explaining the plausible reason of reduced erythropoiesis in the bone-marrow. Furthermore, to identify mechanisms leading to oxidative stress, we established an in-vitro culture system where primary murine bone marrow cells were cultured for 18-20 hours in presence of serum isolated after 6hrs from either PBS treated or BA treated C57BL/6 mice. With the help of confocal microscopy, we observed an increase in mitochondrial superoxide in the cells treated with BA serum; interestingly we have also seen a decrease in Ter 119 population in the cells cultured with BA treated serum implicating that the erythroid cells are dying. To further investigate the downstream players related to the death of erythroid progenitors we are currently investigating the role caspase 1 (a major regulator in pyroptosis) and Gata-1. In conclusion, this study is elucidating some of the mechanisms associated with the anemia triggered by inflammation with the potential to identify new targets and treatments. Disclosures Rivella: Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy; Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 81 We investigated the contribution of macrophages to physiological and pathological conditions in which erythropoietic activity is enhanced. We utilized mouse models of a) anemia by phlebotomy-induced stress erythropoiesis (SE); b) increased erythropoiesis by erythropoietin (Epo) administration; c) Polycythemia Vera (Jak2V617F/+ or PV) and d) beta-thalassemia intermedia (Hbbth3/+ or BTI) in which macrophages were chemically depleted by injection of liposome-clodronate (LC). While chronic injection (up to 3 months) of LC in normal mice had little effect on steady state erythropoiesis, depletion of macrophages severely impaired recovery from anemia following phlebotomy and significantly limited the increase in hematocrit (Htc) in animals treated with Epo. To exclude that this effect was mediated by decreased serum iron parameters, we used mice iron overloaded by dietary means or affected by hemochromatosis (Hfe-KO and Hamp-KO). In these mice, recovery from anemia was still impaired following macrophage depletion, even though serum iron and transferrin saturation levels were elevated and unaffected by LC administration. In vitro studies using both mouse and human primary erythroblasts (EBs) indicated that EBs in S-phase were twice as many compared to EBs cultured in absence of macrophages. The numbers of terminally mature erythroid cells were up to six fold higher in co-culture conditions. Experiments using transwells indicate that direct contact between EBs and macrophages was required to generate this effect. Since our data highlighted an important role of macrophages in enhancing erythropoiesis, we investigated two disorders in which the pool of erythroid progenitor cells is expanded, such as PV and BTI. Chronic administration of LC in PV mice completely reversed splenomegaly and the Htc (P