ALBERT

Description: Introduction: Sickle cell anemia (SCA) is caused by mutations in β-globin that result in the production of the abnormal hemoglobin, HbS, with deleterious effects on erythrocyte shape and life span. Because the propensity of erythrocytes to sickle is inversely proportional to the concentration of HbS in the cell, decreasing the mean cellular hemoglobin concentration (MCHC) represents a potential therapeutic approach. Whereas iron restriction in healthy individuals does not alter MCHC, concomitant iron deficiency has been associated with decreased MCHC in SCA patients. Isolated case reports have linked iron restricted erythropoiesis with decreased hemolysis, increased red cell lifespan, and improvement in certain outcomes in SCA patients. We systematically examined the effects of iron restriction on erythropoietic outcomes in SCA utilizing the Townes murine model to investigate the hypothesis that mice with dietary iron deficiency will demonstrate a decreased MCHC, decreased erythrocyte sickling propensity, and improved anemia compared with mice on an iron sufficient diet. Methods: Townes SCA mice were weaned to diets containing either 20 ppm iron (low) or 48 ppm iron (sufficient) and maintained on those diets until sacrifice at 2 months of age. Blood was collected for complete blood count by submandibular or cardiac puncture. Spleen weight was normalized to body weight for calculation of the splenic index. Red cell deformability, defined by the elongation index (EI), and the oxygen pressure at which sickling occurs (point of sickling) during deoxygenation were characterized by oxygenscan ektacytometry using a laser optical rotational red cell analyzer (Mechatronics, The Netherlands). Results: SCA mice fed a 20 ppm low iron diet demonstrate a significant decrease in MCHC compared to SCA mice fed a 48 ppm iron sufficient diet (17.7+1.1 vs 22.7+5.5 g/dL; p

Description: Nutritional fasting is associated with hypoferremia; however serum iron parameters otherwise differ from those observed with dietary iron deficiency. Activation of the hepcidin gene via the transcription factor CREB-H has been implicated in inducing this hypoferremia. We examined the contribution of the PPARα another hepatocellular transcription factor induced by fasting in this regulation. Six week old male AKR mice (N = 6) were fed diet with 60 ppm Fe ad libitum and fasted for overnight (18 hours). Tissue and blood samples were collected to evaluate iron parameters and gene expression in comparison to fed controls. All mice were provided water ad libitum. In the fasted mice, serum iron (321 and 199 µg/dL, respectively for fed and fasted mice, P = 0.001) as well as transferrin saturation (80% and 64% respectively for fed and fasted mice, P = 0.04) decreased significantly. Fasted mice also had elevated hematocrit (43% and 47%, respectively, for the fed and fasted mice, P = 0.001) in association with weight loss (22g and 18g, respectively, for the fed and fasted mice, P 〈 0.01), consistent with hemoconcentration. In addition, 18h fasting induced a significant increase in hepatic (735 and 953 µg/g dry, respectively for the fed and fasted mice, P 〈 0.05) and splenic non-heme iron content (414 and 488 µg/g dry, respectively for the fed and fasted mice, P 〈 0.001), and elevated hepatic hamp1 mRNA levels (4-fold increase from the fed group, P 〈 0.001). To elucidate the mechanism of the Hamp1up-regulation, we measured liver Creb-h and pparα mRNA levels, and observed elevated Creb-h (by 4-fold, P 〈 0.001) and ppara mRNA levels (by 3-fold, P 〈 0.001) in the fasted mice. To further elucidate the roles of CREB-H, and PPARα in the regulation of Hamp1 during fasting, we performed fasting-refeeding studies in 10-12 week old male C57BL/6 mice. These mice (N = 4-6) were fed with chow ad libitum, fasted overnight, or fasted overnight then re-fed for 4 hours. Fasting at this age also induced increased in liver pparα, creb-h, and hamp1 mRNA levels (by 2.5-, 3- and 6-fold, respectively), and refeeding over this timeframe was associated with decreases in pparα, creb-h, and hamp1 to levels that are similar to fed mice, even though the serum and tissue iron levels were not restored. To determine if PPARα contributes to the regulation of hepcidin, we examined the fasting response of PPARα-/- mice. The fasting-associated increases in expression of hamp1 and creb-h were dampened in the PPARα-/- mice. In the WT mice, fasting induced a hamp1 increase of 2.6-fold (P = 0.002), whereas in the PPARα-/- mice, the expression was unchanged (0.8-fold, P = 0.11). Similarly, fasting induced a creb-h increase of 2.8-fold in the WT mice (P = 0.0003) but a 1.1-fold increase in the PPARα-/- mice (P = 0.004). The magnitude of change in hamp1 (P 〈 0.05) and in creb-h (P 〈 0.001) expression between WT and PPARα-/- mice were statistically significant. These findings indicate that PPARα contributes to creb-h and hamp1 up-regulation during fasting-induced changes in iron metabolism. Disclosures No relevant conflicts of interest to declare.

Description: Iron overload causes morbidity and mortality in patients with β-thalassemia. Transfusion independent patients develop iron overload from increased dietary iron absorption, implicating inappropriately low hepcidin and supporting the therapeutic potential of approaches to increase hepcidin. Relatively low liver hepcidin mRNA expression is also characteristic of mouse models of β-thalassemia, and a recently identified erythroid factor, erythroferrone, has been implicated in hepcidin suppression in thalassemia. We have previously shown that exogenous apo-transferrin injections result in relatively iron restricted erythropoiesis, ameliorate ineffective erythropoiesis, and increase hepcidin expression in Hbbth1/th1 β-thalassemia intermedia (thalassemic) mice. We now explore the effect of exogenous apo-transferrin on signaling pathways (i.e. Smad and Erk) and circulating parameters thought to participate in hepcidin regulation in vivo and in vitro in wild type (WT) and thalassemic mice. Our results demonstrate that apo-transferrin injection increase both serum hepcidin concentration (603 vs. 306 ng/ml, n=13-24 per group, P

Description: Transferrin receptor 2 (TfR2) is a membrane glycoprotein that mediates cellular iron uptake from holotransferrin. Homozygous mutations of this gene cause one form of hereditary hemochromatosis in humans. We recently reported that homozygous TfR2(Y245X) mutant mice, which correspond to the TfR2(Y250X) mutation in humans, showed a phenotype similar to hereditary hemochromatosis. In this study, we further analyzed the phenotype as well as iron-related gene expression in these mice by comparing the TfR2-mutant and wild-type siblings. Northern blot analyses showed that the levels of expression of hepcidin mRNA in the liver were generally lower, whereas those of duodenal DMT1, the main transporter for uptake of dietary iron, were higher in the TfR2-mutant mice as compared to the wild-type siblings. Expression of hepcidin mRNA in the TfR2 mutant mice remained low even after intraperitoneal iron loading. In isolated hepatocytes from both wild-type and TfR2 mutant mice, interleukin-6 and lipopolysaccharide each induced expression of hepcidin mRNA. These results suggest that up-regulation of hepcidin expression by inflammatory stimuli is independent of TfR2 and that TfR2 is upstream of hepcidin in the regulatory pathway of body iron homeostasis. (Blood. 2005;105:376-381)

Description: Key PointsInvestigation of the iron-restrictive effect of minihepcidin peptides in the treatment of β-thalassemia and polycythemia vera.

Description: Key Points Apotransferrin decreases TfR1 expression and alters TfR1 trafficking to normalize enucleation in β-thalassemic erythroid precursors. Decreased TfR1 upregulates hepcidin in an iron- and ERFE-independent manner, resulting in iron-restricted β-thalassemic erythropoiesis.

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: β-thalassemia is an inherited blood disorder caused by reduced or absence of β-globin expression which results in imbalanced globin synthesis, ineffective erythropoiesis, and anemia. How the imbalance between α- and β-globin results in ineffective erythropoiesis, if apoptosis or dysfunctional differentiation of erythroid precursors results in ineffective erythropoiesis, and whether disrupted iron regulation and / or iron overload in β-thalassemia is directly involved in the pathophysiology of ineffective erythropoiesis is incompletely understood. Iron is critical for hemoglobin synthesis and erythropoiesis is dependent on transferrin (Tf) bound iron. Tf functions as the main iron transporter in circulation, where it exists in three forms: as iron-free apo-transferrin (apoTf), monoferric Tf, or diferric Tf (holoTf). Typically, iron is bound to 30% of all Tf binding sites in circulation at which point monoferric Tf is found in the highest concentration relative to holoTf. . We have previously shown that exogenous apoTf ameliorates anemia in a mouse model of β-thalassemia intermedia (th1/th1), resulting in reduced splenomegaly, reticulocytosis, and α-globin precipitation on circulatory red blood cells (RBC). We also observe a decrease in mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV), serum iron, Tf saturation, together suggesting that relative iron deficiency improves iron metabolism and ineffective erythropoiesis in apoTf-treated th1/th1 mice. We hypothesize that exogenous apoTf decreases cytosolic iron and heme as a consequence of increased monoferric Tf which results in less iron entering cells via Tf:TfR1 binding. Our current data reveals that in vitro incubation of purified apoTf and holoTf at 37C results in the formation of monoferric Tf, and injection of wild-type (WT) mice with a single intraperitoneal dose of apoTf (10mg) decreases holoTf (P=0.01) and increases monoferric Tf (P=0.02) in the serum 6 hours after injection. Using both calcium mobilization and anti-Tf antibodies in flow cytometry, we demonstrate that apoTf results in no TfR1 binding relative to holoTf in CHO cells (P