ABSTRACT:Anemia of inflammation (AI) is the second most common form of anemia and is prevalent in patients with chronic inflammatory states, such as infection, autoimmunity, and cancer. Interleukin 6 (IL-6) is well-known to induce the iron-sequestering hormone hepcidin, which results in iron-restricted anemia. The contributions of other proinflammatory cytokines, such as tumor necrosis factor-α (TNFα) and interferon gamma (IFNγ), are less understood in the pathophysiology of AI. This study investigated the role of TNFα in a mouse model of AI by administering heat-killed Brucella abortus (HKBA) to germ line TNFα knockout (KO) mice. We hypothesized that TNFα possessed an important role in restoring steady-state erythropoiesis after inflammatory insult. TNFαKO injected with HKBA displayed a chronic anemia, with elevated proinflammatory IL12p40 and IFNγ cytokines that did not resolve. However, IFNγKO and TNFαKO/FNγKO double knockout mice showed reduced inflammation and anemia following HKBA administration. Because IFNγKO displayed normal serum TNFα and IL12p40 levels, we hypothesized that the persistent anemia was IFNγ induced and TNFα was necessary for AI cessation. However, treatment with recombinant TNFα (rTNFα) accelerated death, while reducing IFNγ using an anti-IFNγ antibody (Ab) only briefly improved anemia. Only the combination of both the Ab and rTNFα together reversed the hyperinflammatory phenotype, restored erythropoiesis, and prevented death of TNFαKO + HKBA mice. Our data provide compelling evidence for an anti-inflammatory role of TNFα that is necessary for the restoration of erythropoiesis and mitigation of proinflammatory IFNγ action in a mouse model of AI.

Anemia of inflammation (AI) is the second most prevalent form of anemia, and is common in patients with chronic inflammatory states, such as infection, autoimmunity, and cancer. There are no targeted treatments available for AI, strategies typically focus on treating the underlying disease. Inhibiting tumor necrosis factor-α (TNFα) has become the gold standard for treating autoimmune disorders. However, some patients show exacerbations or onsets of new autoimmune conditions following treatment. We became interested in the effects of TNFα on erythropoiesis due to reports of TNFα blocking antibodies (Ab) improving anemia as a secondary treatment outcome. We started our studies using a well-established model of AI induced by injection of heat-killed Brucella Abortus (BA) in germline TNFα knockout (TNFαKO) mice. Our findings show that TNFαKO+BA mice developed a macrocytic hyperchromic anemia, leukocytosis, and abnormally increased myeloid and lymphocytes populations in the bone marrow (BM) and spleens, which resulted in death after 10-weeks. Serum cytokine analysis of TNFαKO+BA mice displayed sustained elevations of interleukin (IL)12p40 and interferon-γ (IFNγ) levels. Given the reports of improvements in anemia in patients treated with TNFα blocking agents, these surprising findings made us question whether TNFα played an unknown anti-inflammatory role which impacted erythropoiesis.IFNγ's role in RBC lifespan, erythrophagocytosis, and BM failure is well-established. We hypothesized that TNFα played an important anti-inflammatory role in modulating IFNγ. IFNγKO+BA mice showed normal TNFα serum levels, a reduced inflammation profile, normal red blood cell (RBC) counts and minimal perturbations to erythropoiesis at time points tested. Additionally, a TNFαKO/IFNγKO double knock-out (DKO) mouse line was generated and challenged with BA to test if lack of IFNγ would correct the TNFαKO+BA phenotype. Indeed, DKO+BA mice had a phenotype closer to that of IFNγKO+BA and WT+BA than TNFαKO+BA. We tested if administration of recombinant TNFα (rTNFα) would correct the chronic AI phenotype in TNFαKO+BA. TNFαKO+BA treated with rTNFα died shortly after administration. We also tested if reduction of IFNγ by an anti-IFNγ Ab would rescue TNFαKO+BA mice. We found that treatment with anti-IFNγ Ab partially corrected the anemia phenotype but reverted after 4 weeks. However, rTNFα in combination with anti-IFNγ Ab reversed the hyper-inflammatory phenotype, rescued erythropoiesis, and prevented death in TNFαKO+BA mice. We next sought to identify the cause of death in TNFαKO+BA. Chronic inflammation is well known to increase proliferative stress of hematopoietic stem cells (HSC) and contribute to their accelerated exhaustion. Increased IFNγ during chronic inflammatory stress negatively affects HSC homeostasis by skewing HSC towards differentiation, impeding self-renewal and resulting in HSC exhaustion. We analyzed the HSC compartment of TNFαKO+BA at 8 weeks and found a dramatic increases in HSCs, myeloid and the lymphoid compartment, suggesting that HSCs in TNFαKO+BA exhibit elevated proliferation and differentiation activities. To assess whether the elevated HSC cycling phenotype in TNFαKO+BA leads to HSC exhaustion, we transplanted whole BM from untreated-WT, untreated-TNFαKO, WT+BA (8 weeks) or TNFαKO+BA (8 weeks) into myeloblated CD45.1 mice. Our preliminary data showed that recipient mice transplanted with whole BM of TNFαKO+BA died or had to be sacrificed due to low red blood cell (RBC) counts by 4-weeks post BMT, while the other groups had normal RBC levels. Interestingly, CD45.1 recipients with donor TNFαKO+BA cells showed no splenomegaly. Additionally, untreated-TNFαKO whole BM CD45.1 recipients displayed elevations in lymphocyte counts a 4-week post BMT compared to controls. These experiments are ongoing. TNFα has been demonstrated to provide essential pro-survival signals to HSCs for the resolution of inflammation. We are currently working towards understanding the direct effects of TNFα on erythroid populations by focusing on the pathways regulated by the TNFα receptors during inflammation. Our work supports an essential role for TNFα in resolving the inflammatory response, characterizes a new model to investigate the role of TNFα in the etiology of AI, and provides potential clues for exacerbations in patients treated with TNFα inhibitors.

In this study, we investigated the role of TNFα in the development of anemia of inflammation (AI) in a TNFα knockout (KO) mouse model of AI, which is induced via one intraperitoneal (i.p.) injection of heat-killed Brucella Abortus (BA) (Gardenghi et al., 2014; Kim et al., 2014). Due to reports of amelioration of anemia in patients with autoimmune disorders as secondary outcomes (Kalliolias et al., 2016) when treated with anti-TNFα drugs, we hypothesized that TNFαKO mice would show a less severe form of AI when challenged with BA. Our results were both unexpected and astonishing. TNFαKO-BA mice developed leukocytosis, with elevated macrophages and T-lymphocytes, and an irreversible macrocytic, hyperchromic anemia, which resulted in death after 10 weeks. Serum analysis showed that erythropoietin, iron and hepcidin were elevated in TNFαKO-BA, which ruled out iron-restriction as the cause for the persistent anemia. However, serum cytokine measurements of TNFαKO mice at 4 weeks showed continual elevation of interleukin (IL)-12p40 and interferon-γ (IFNγ) compared to WT-BA controls. Chronic inflammation is well known to increase proliferative stress of hematopoietic stem cells (HSC) and contribute to their accelerated exhaustion. Increased IFNγ during chronic inflammatory stress negatively affects HSC homeostasis by skewing HSC towards differentiation, impeding self-renewal and resulting in HSC exhaustion (Morales-Matilla and King, 2018). In 2019, Yamashita & Passegue, showed that TNFα plays a protective role in HSC regeneration during inflammatory stress. Therefore, we analyzed the HSC compartment of TNFαKO -PBS or -BA treated mice at 8 weeks. We found a dramatic increase in HSCs and common lymphoid progenitors (CLP), suggesting that TNFαKO-BA mice might die due to bone marrow failure caused by chronic inflammation. We questioned if TNFα served an anti-inflammatory role in a feedback mechanism between IL-12p40 and IFNγ secreting cells. In the absence of TNFα, we hypothesized, HSCs would be unable to return to quiescence and would be skewed towards the CLP lineage, resulting in pronounced anemia due to decreased erythroid progenitors because of this skewing. To test if concurrent loss of IFNγ would correct the observed phenotype, we crossed TNFαKO with IFNγKO mice (DKO). Indeed, IFNγKO and DKO mice challenged with BA showed a mild anemic phenotype compared to WT-BA and TNFαKO-BA. Additionally, serum levels of IL-12p40 were normalized by 4 weeks in IFNγKO-BA and DKO-BA compared to TNFαKO-BA mice. Analysis of livers and SPLs of BA treated mice by immunohistochemistry using an anti-CD3 antibody shows disorganization of the white pulp in the SPL and infiltration of T-Lymphocytes in livers of TNFαKO-BA but not in WT-BA, IFNγKO-BA or DKO-BA animals at 8 weeks. Lastly, retro-orbital injection of TNFαKO-BA mice between 2-8 weeks with an anti-IFNγ antibody (0.25mg) did not correct the phenotype. However, combination treatment with an anti-IFNγ antibody (0.25mg) in combination with recombinant TNFα (0.02mg), rescued the anemia phenotype. Current experiments are assessing the effects of TNFα and anti-IFNγ antibody combination treatment on the immune and HSC compartments of TNFαKO-BA mice. Currently, treatments that block TNFα are central for the management of autoimmune diseases. However, some patients do not respond to TNFα inhibitor treatment. Others show exacerbation of a pre-existing autoimmune disease or the onset of a new autoimmune condition following anti-TNFα treatment (Salomon 2021). This finding in patients reveals an anti-inflammatory role most likely explained by the downstream effects of the TNF receptor 2, which activate pro-survival signals via the NFkB signaling pathway (Salomon 2021). In this study we demonstrate that in the absence of TNFα, injection with BA induces uncontrolled elevation of IL12p40 and IFNγ, producing a phenotype which included lymphocytic infiltration, thymic atrophy and possible HSC exhaustion. Administering anti-IFNγ antibody alone was not sufficient to rescue the TNFαKO-BA phenotype. Administration of TNFα was essential in rescuing TNFαKO-BA mice. This work supports the notion that both inflammatory and regulatory roles of TNFα and associated pathways need to be better understood to develop better and safer anti-TNFα therapies to treat autoimmune disorders.

Abstract Anemia of inflammation (AI) 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 is an inflammatory cytokine which also limits iron absorption by inducing HAMP, which promotes the degradation of the iron exporter ferroportin. We hypothesized that knocking down both HAMP and IL6 simultaneously will help us to understand if their mode of action in AI is uniquely limited to iron absorption and erythroid iron intake or if they also have independent roles. Henceforth, we generated IL6/HampKO (DKO) mice and, unexpectedly, observed that IL6KO mice showed the best recovery in bone marrow (BM) erythropoiesis (using flow cytometry analysis and looking at the absolute number of erythroid progenitors) after BA administration when compared to wild type (WT), HampKO and DKO mice. The best differences were observed at 14 days post BA administration. In contrast, the extramedullary erythropoiesis in the spleen was more pronounced in HampKO and DKO mice compared to WT and IL6KO animals, indicating that the mechanism impairing erythropoiesis in the BM did not affect erythroid progenitors in the spleen. These observations suggest that HAMP and IL6 proteins contribute independently to AI, with IL6 having some effect on the erythropoiesis in the BM independent from the IL6-HAMP axis leading to iron restriction. Furthermore, these observations raised the question why both HampKO and DKO mice showed reduced BM erythropoiesis compared to IL6KO animals. We investigated inflammatory cytokines and altered iron parameters as potential mediators of impaired erythropoiesis. We compared several inflammatory cytokines, including IL6, TNFa and INFg following BA administration: cytokine levels were elevated 6 hrs, reduced 48hrs after BA administration and moderately increased again two weeks later. Interestingly, among all the cytokines the levels of IL1b were significantly attenuated in IL6KO mice at day 14 compared to WT and HampKO animals. Moreover, transferrin saturation and NTBI levels were higher in HampKO and DKO animals compared to IL6KO mice. These observations strongly suggested that BM erythropoiesis is more sensitive to inflammatory insult in presence of an excess of iron, while extramedullary erythropoiesis is mildly affected and can eventually thrive under supra-physiological transferrin saturation levels. To test if increased iron affects BM erythropoiesis in presence of inflammation, we treated both WT and IL6KO mice with combination of iron dextran and BA. Both WT and IL6KO mice were treated with a combination of BA and iron at day 0 followed by alternate day of iron injections showed the poorest erythropoiesis in the BM and became rapidly sick, although the effect was significantly more pronounced in WT animals, as suggested by their survival curve. Since mycobacterium infections lead to NLPR3 inflammasome activation and Caspase1 upregulation (Marim et al. Semin Immunopathology 2017), we investigated how erythroid progenitors were affected. By flow cytometry analyses, we observed a significantly higher upregulation of the Caspase1 protein in WT and DKO mice compared to IL6KO animals. This was also reproduced by culturing WT or IL6KO BM progenitor erythroid cells in presence of mouse serum derived from WT or IL6KO mice treated with BA. Most importantly, IL6KO mice treated with BA and iron showed the highest levels of Caspase1 compared to only BA treated IL6KO mice, indicating that excess of iron abrogates the beneficial effect of IL6 deficiency on erythropoiesis under conditions of AI. Furthermore, using flow cytometry, we observed in WT mice treated with BA or BA and iron a significant increase in mitochondrial mass, which is an indicator of mitochondrial stress. The mitochondrial mass was reduced in IL6KO mice treated with BA, but again increased in IL6KO mice treated with BA and iron. We have also observed an increase of mitochondrial superoxide by confocal microscopy in WT mice compared to IL6KO mice treated with BA. Altogether, these data support a model where inflammation in presence of an excess of iron impairs BM erythropoiesis through mechanisms at least in part mediated by Caspase1 and mitochondrial dysfunction, while iron excess itself is sufficient to boost extramedullary erythropoiesis to compensate and sustain RBC production. Disclosures Vinchi: PharmaNutra: Research Funding; Vifor Pharma: Research Funding; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding. Rivella: Ionis Pharmaceuticals: Consultancy; Meira GTx: Consultancy.

Anemia of Inflammation (AI) is prevalent in patients with chronic inflammatory states, such as infection, autoimmunity, or cancer. Induced expression of hepcidin by pro-inflammatory cytokines results in iron-restricted anemia. In particular, abnormally elevated levels of the cytokine Tumor Necrosis Factor-α (TNFα) is a hallmark of AI, however its contribution to the pathophysiology of AI is not well understood.

Minihepcidins are hepcidin agonists that have been previously shown to reverse iron overload and improve erythropoiesis in mice affected by non-transfusion-dependent thalassemia. Given the extreme anemia that occurred with the previous model of transfusion-dependent thalassemia, that model was inadequate for investigating whether minihepcidins can improve red blood cell quality, lifespan and ineffective erythropoiesis. To overcome this limitation, we generated a new murine model of transfusion-dependent thalassemia with severe anemia and splenomegaly, but sufficient red cells and hemoglobin production to test the effect of minihepcidins. Furthermore, this new model demonstrates cardiac iron overload for the first time. In the absence of transfusions, minihepcidins improved red blood cell morphology and lifespan as well as ineffective erythropoiesis. Administration of a minihepcidin in combination with chronic red blood cell transfusion further improved the ineffective erythropoiesis and splenomegaly and reversed cardiac iron overload. These studies indicate that drugs such as minihepcidins have therapeutic potential for patients with transfusion-dependent thalassemia.

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.

Stress erythropoiesis (SE) is characterized by an imbalance in erythroid proliferation and differentiation under increased demands of erythrocyte generation and tissue oxygenation. β-thalassemia represents a chronic state of SE, called ineffective erythropoiesis (IE), exhibiting an expansion of erythroid-progenitor pool and deposition of alpha chains on erythrocyte membranes, causing cell death and anemia. Concurrently, there is a decrease in hepcidin expression and a subsequent state of iron overload. There are substantial investigative efforts to target increased iron absorption under IE. There are also avenues for targeting cell contact and signaling within erythroblastic islands under SE, for therapeutic benefits.

Inflammatory states seen in infections and chronic disorders are often characterized by a condition called anemia of Inflammation (AI). Using a mouse model of AI generated by a single injection of the heat killed pathogen Brucella abortus (BA), we have previously shown that mice lacking IL6 (IL6 -KO mice) exhibited protected erythropoiesis in the bone marrow (BM) and a faster recovery from anemia compared to controls. To study the mechanism of IL6 mediated improved erythropoiesis under AI, we investigated erythroid recovery in WT and IL6 -KO mice injected with BA. 72 hours following BA administration, both genotypes showed impaired BM erythropoiesis associated by a surge in inflammatory cytokines such as IFNγ and TNFα and a concurrent increase in mitochondrial ROS (reactive oxygen species), specifically superoxide (SO) in erythroid progenitors. Cytokines levels were normalized after 24 hours. However, during the second phase of erythroid recovery (10-14 days following BA treatment), mice showed a second surge of inflammatory cytokines. During this phase, IL6 -KO mice showed significantly improved BM erythropoiesis and normalization of ROS levels. We analyzed SO levels by Mitosox red, generic ROS production using the chloromethyl derivative of 2′,7′-dichlorofluorescin diacetate and total protein oxidation (by looking at total protein carbonylation) by Western blot. These results were in sharp contrast to WT animals which continued to show upregulated ROS, total protein oxidation and slackened erythroid recovery.

Anemia of inflammation (AI) is a hallmark of a multitude of disorders including infections, autoimmune diseases and malignancies. The pathophysiology of AI is currently under investigation. While the hematological aspect of AI and the involvement of cytokines such as IL6, IL1α, IL1 β has been subject to extensive study, the inflammatory and immunological arm has only recently begun to be explored. This involves a complex interplay of cytokine mediators both pro and anti-inflammatory, mediating a broader innate and ultimately adaptive immune response that could potentially be linked to the hematological deficiencies seen under AI. In particular, highly elevated levels of the cytokine TNF a have been noted in inflammatory conditions, including an established acute mouse model of AI induced by a single injection of the heat killed pathogen Brucella abortus (BA). The latter results observed by us prompted us to furtherinvestigate the role of this cytokine in BA induced AI.

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).

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/th1 and Hbbth2/+. These mice harbor alternative mutations so that the synthesis of the mouse b-globin genes is different in each model. Hbbth1/th2 and Hbbth1/th3 pups 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/th2 FLCs, 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/th2 and Hbbth1/th3 models. 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.

Alternative splicing has emerged as a vital way to expand the functional repertoire of a set number of mammalian genes. For example, such changes can dramatically alter the function and cellular localization of transcription factors. With this in mind, we addressed whether EKLF/KLF1 mRNA, coding for a transcription factor that plays a critical role in erythropoietic gene regulation, is alternatively spliced. We find that EKLF mRNA undergoes exon skipping only in primary tissues and that this splice variant (SV) remains at a very low level in both embryonic and adult erythroid cells, as well as during terminal differentiation. The resultant protein is truncated and partially encodes a non erythroid Kruppel-like factor amino acid sequence. Its overexpression can alter full-length erythroid Kriippel-like factor function at selected promoters. We discuss these results in the context of stress and with respect to recent global studies on the role of alternative splicing during terminal erythroid differentiation. Copyright (C) 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc.

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.

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.

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 (Hbb th3/+ ) 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 (Hbb th3/+ , Itgb 1fl/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.

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.