Epeleuton, a novel synthetic ω-3 fatty acid, reduces hypoxia/ reperfusion stress in a mouse model of sickle cell disease

Inflammatory vasculopathy is critical in sickle cell disease (SCD)-associated organ damage. An imbalance between pro-inflammatory and pro-resolving mechanisms in response to different triggers such as hypoxia/reoxygenation or infections has been proposed to contribute to the progression of SCD. Administration of specialized pro-resolving lipid mediators may provide an effective therapeutic strategy to target inflammatory vasculopathy and to modulate inflammatory response. Epeleuton (15 hydroxy eicosapentaenoic acid ethyl ester) is a novel, orally administered, second-generation ω-3 fatty acid with a favorable clinical safety profile. In this study we show that epeleuton re-programs the lipidomic pattern of target organs for SCD towards a pro-resolving pattern. This protects against systemic and local inflammatory responses and improves red cell features, resulting in reduced hemolysis and sickling compared with that in vehicle-treated SCD mice. In addition, epeleuton prevents hypoxia/reoxygenation-induced activation of nuclear factor-κB with downregulation of the NLRP3 inflammasome in lung, kidney, and liver. This was associated with downregulation of markers of vascular activation in epeleuton-treated SCD mice when compared to vehicle-treated animals. Collectively our data support the potential therapeutic utility of epeleuton and provide the rationale for the design of clinical trials to evaluate the efficacy of epeleuton in patients with SCD

Mitapivat Improves Transfusion Burden and Reduces Iron Overload in Thalassemic Mice

Abstract Mitapivat, an oral activator of pyruvate kinase (PK), was recently shown to improve b-thalassemic anemia with a reduction of ineffective erythropoiesis and an amelioration of b-thalassemic red cell features in a mouse model for b-thalassemia (Hbb 3th/+ mice).). These changes were also associated with a beneficial effect on iron homeostasis by modulation of duodenal DMT1 expression (Matte A et al JCI 2021). Two clinical studies have shown improvement of anemia and ineffective erythropoiesis with mitapivat treatment in patients with non-transfusion-dependent (NTD) thalassemia (Kuo et al. EHA 2021). Based on these results, Phase 3 studies in both NTD and TD thalassemia are currently on going. The objective of this preclinical study was to determine if treatment with mitapivat affects the length between transfusion of red blood cells (RBCs) and the liver iron concentration (LIC). Using a previously established murine model of RBCs transfusions (Park Y et al Blood 2020), in Hbb 3th/+ mice, we used Hb 10.5 g/dL as threshold for RBCs transfusion, with washed RBCs, at 40% Hct (400 uL total volume infused). The animals were divided into two groups: vehicle and mitapivat (50mg/Kg by gavage BID for up to 61 days).The length of the interval between transfusions increased in mitapivat treated compared to vehicle treated animals (transfusion interval: 13.8±1.0 days vs vehicle 10.5±1.0 days respectively n=4 and n=3). In both groups, the transfusion regimen induced a significant reduction in spleen weight/mouse weight ratio and in extramedullary erythropoiesis. We also found a significant reduction in liver iron content (LIC) in mitapivat treated compared to vehicle treated animals. We then evaluated the effects of mitapivat in combination with iron chelation using deferiprone (DFP,1.25 mg/mL, drinking water). Casu et al. have previously shown in the same mouse model for β-thal that DFP did not affect erythropoiesis. In the β-thal mice, we did not find negative effects on hematologic parameters when mitapivat (50 mg/Kg/d by gavage BID) was co-administrated with DFP for 28 days. LIC was reduced in mitapivat treated mice and in mitapivat +DFP treated β-thal mice was further decreased compared to vehicle treated animals. This allowed us to reduce DFP dosage from 1.25 to 0.8 mg/mL in mitapivat treated β-thal mice. These data show that in mouse model of transfused β-thalassemia, mitapivat increases the time interval between transfusions, reduces transfusion burden and allows a reduction of the dosing iron chelation with DFP. Thus, mitapivat might represent an interesting option in transfusion dependent β-thalassemic patients, being transfusion burden still an unmet need in this patient population. Disclosures Kosinski: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Dang: Agios Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. De Franceschi: F. Hoffmann-La Roche Ltd: Consultancy. </jats:sec

Resolvin D1 and Resolvin D2 Protect Against Hypoxia/Reoxygenation Induced Lung and Kidney Damage in a Sickle Cell Mouse Model of Acute Vaso-Occlusive Crisis

Abstract Sickle cell disease (SCD) is characterized by hemolytic anemia in association with acute and chronic life-threatening clinical complications. Acute vaso-occlusive crisis (VOCs) are the main cause of hospitalization for SCD patients. In VOCs, amplified inflammatory response plays a key role in acute organ damage. Pro-Resolving lipid mediators such as resolvins (Rv) accelerate resolution of acute inflammation in different models, indicating that stimulation of endogenous resolution of inflammatory processes may be an additional strategy in limiting tissue damage. Recent data suggest beneficial effects of Rvs in hypoxia/reoxygenation (H/R) related tissue injury. Here, we study the effects of Rvs on a model of acute VOCs using humanized SCD mice (Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB*)Tow). We treated SCD and control healthy mice (AA, Hbatm1(HBA)Tow Hbbtm3(HBG1,HBB)Tow) (n =6-7 animals in each group) with RvD1, 7S, 8R, 17S- trihydroxy-docosa-4Z, 9E, 11E, 13Z, 15E, 19Z-hexaenoic acid, 17R- RvD1, 7S, 8R, 17R- trihydroxy-docosa-4Z, 9E, 11E, 13Z, 15E, 19Z-hexaenoic acid; RvD2, 7S, 16R, 17S-trihydroxy-docosa-4Z, 8E, 10Z, 12E, 14E, 19Z-hexaenoic acid. Mice were treated with RvD1 and RvD2 at the dose of 2.5 ug/Kg by gavage 1 hour (hr) before H/R stress (10 hrs 8% oxygen followed by 3 hrs reoxygenation), which we have used in the past to mimic acute VOCs. We found that RvD1 and RvD2 significantly reduced the H/R-induced (i) increase in neutrophil count; (ii) lung inflammatory cell infiltrate, mucus and thrombi formation; (iii) glomerular inflammatory cell infiltration, glomerular sclerosis and thrombi formation. In the lung of H/R SCD mice, RvD1 prevented the H/R induced up-regulation of (i) cytokines such as MCP2, IL-6 and ET-1; (ii) vascular endothelial activation markers (VCAM-1 and ICAM-1; (iii) cytoprotective systems such as Prx6 and HO-1. In the kidney of H/R SCD mice, RvD1 significantly reduced H/R induced expression of IL-6 and ET-1 as well as HO-1. Our data indicate that RvD1 and RvD2 modulating inflammatory responses related to H/R in SCD, protect sickle cell target organs, and foster resolution. Thus, RvD1 and RvD2 might represent a novel therapeutic approach for acute VOCs in SCD. Disclosures No relevant conflicts of interest to declare. </jats:sec

Fyn Kinase Is Involved in EPO Receptor Signaling and Is Required to Harmonize the Response to Oxidation

Abstract Erythropoiesis is a complex multistep process during which committed erythroid progenitors undergo terminal differentiation to produce circulating mature red cells. Erythroid differentiation is characterized by the production of reactive oxygen species (ROS) both in response to erythropoietin (EPO) and to the large amount of iron imported into the cells for heme biosynthesis. During erythropoiesis, ROS might function as second messenger by modulating intracellular signaling pathways. Fyn, an Src kinase, has been previously reported to participate in signaling pathways in response to ROS in various cell types. Here, we explore the potential contribution of Fyn to normal and stress erythropoiesis by studying 2-4 months-old Fyn knockout mouse strain (Fyn-/-) and C57B6/2J as wild-type controls. Fyn-/- mice showed a mild compensated microcytic anemia associated with signs of dyserythropoiesis. Increased ROS levels and Annexin-V+ cells were presented in all Fyn-/- erythroblast subpopulations compared to wild-type, suggesting a possible reduction in the efficiency of erythropoietin (EPO) signaling pathway in the absence of Fyn. Indeed, in Fyn-/- erythroblasts we observed a reduction in Tyr-phosphorylation state of EPO-R associated with a compensatory activation of Jak2 without major change in Lyn activity. A reduction in STAT5 activation resulting in down-regulation of Cish, a known direct STAT5 target gene, was noted in Fyn-/- erythroblasts. This was paralleled by a reduction in GATA1 and increased HSP70 nuclear translocation compared to wild type, supporting a higher cellular pro-oxidant environment in the absence of Fyn. Using the vitro cell forming colony unit assay, we found a lower in CFU-E and BFU-E cells production, which once again was associated with decreased activation of EPO mediated cascade in the absence of Fyn. To explore the possible role of Fyn in stress erythropoiesis, mice were treated with either phenylhydrazine (PHZ) or doxorubicin (Doxo). Fyn-/- mice showed prolonged anemia after either PHZ or Doxo treatment with a delayed hematologic recovery compared to wild-type animals. When we analyzed the expression of a battery of ARE-genes related to oxidative response such as catalase, Gpx, heme-oxygenase 1 and peroxiredoxin-2, we noted up-regulated expression of these genes in sorted Fyn-/- erythroblasts compared to wild-type cells. In agreement, we observed increased activation of the redox-sensitive transcriptional factor Nrf2 targeting ARE-genes, whose regulation has been previously linked to Fyn. In fact, Nrf2 is switched-off by Fyn, ubiquitylated and delivered to the autophagosome by the p62 cargo protein. In Fyn-/- sorted erythroblasts, we observed (i) accumulation of p62 in large clusters; and (ii) reduction of Nrf2-p62 complex compared to wild-type cells. To address the question whether the perturbation of Nrf2-p62 system results in impairment of autophagy in the absence of Fyn, we used Lysotrack to explore late phases of autophagy. Lysosomal progression was defective in Fyn-/- reticulocytes and it was associated with accumulation of p62 during in vitro reticulocyte maturation. These data indicate that the absence of Fyn blocks the Nrf2 post-induction response to oxidation, resulting in impaired autophagy. To validate our working hypothesis, we treated Fyn-/- mice with Rapamycin, an inducer of autophagy. In Fyn-/- mice, Rapamycin treatment resulted in decrease dyserythropoiesis, ROS levels and Annexin V+ cells, associated with reduction in accumulation of p62 in Fyn-/- erythroblasts. As a proof of concept, we treated both mouse strains with PHZ with or without Rapamycin. This latter worsened PHZ induced acute anemia in wild-type mice but not in Fyn-/- animals. Collectively, our data enabled us to document a novel role for Fyn in erythropoiesis, contributing to EPO-R activation and harmonizing the Nrf2-p62 adaptive cellular response against oxidation during normal and more importantly in stress erythropoiesis. Disclosures No relevant conflicts of interest to declare. </jats:sec

Effects of hypoxia–reoxygenation stimuli on renal redox status and nuclear factor erythroid 2‐related factor 2 pathway in sickle cell SAD mice

International audienc

Identification and characterization of human CD34⁺ and CD34^dim/- neutrophil-committed progenitors

ABSTRACTWe report the identification of human CD66b−CD64dimCD115− neutrophil-committed progenitors within SSClowCD45dimCD34+ and CD34dim/− bone marrow cells, that we named neutrophil myeloblast (NMs). CD34+ and CD34dim/−NMs resulted as either CD45RA+ or CD45RA−, with CD34+CD45RA−NMs found as selectively expanded in chronic-phase chronic myeloid leukemia patients. By scRNA-seq experiments, CD34+ and CD34dim/−NMs were found to consist of combinations of four cell clusters, characterized by different maturation stages and distributed along two differentiation routes. Cell clusters were identified by neutrophil-specific gene profiles, one of them associated to an interferon-stimulated gene (ISG) signature, hence supporting recently identified expansions of mature neutrophil subsets expressing ISGs in blood of diseased individuals. Altogether, our data shed light on the very early phases of neutrophil ontogeny.</jats:p

Dietary Omega-3 Fatty Acid Supplementation Improves Sickle Cell Bone Disease By Affecting Osteoblastogenesis and Adipogenesis

Abstract Sickle bone disease (SBD) is a severe and invalidating complication related to recurrent bone vaso-occlusive events affecting patients with sickle cell disease (SCD). In a humanized mouse model for SCD, we previously showed that SBD is sustained by impaired osteoblast function and increased osteoclast activity associated with local up-regulation of pro-inflammatory cytokines and anti-oxidant systems (Dalle Carbonare L., Blood 2015;126:2320-2328). Growing evidence supports a role of ω-3 fatty acid supplementation in improving bone homeostasis (Fallon EM, et al J Surg Res 2014;191:148-155.). A diet enriched with ω-3 fatty acids beneficially impacts SCD inflammatory vasculopathy, and blunts the acute and chronic SCD-related organ damage in humanized SCD mice (Kalish BT et al Haematologica 2015;100:870-880). Here, we sought to compare the dietary effects of ω-6 (soybean oil-based, SD)- vs ω-3 (fish oil-based, FD)-enriched diets on SBD in SCD mice (Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB*)Tow). Treated SCD and control healthy mice (AA, Hbatm1(HBA)Tow Hbbtm3(HBG1,HBB)Tow) (n=6 animals in each group) were exposed to recurrent hypoxia/reoxygenation (rH/R) stress, which closely mimics SBD natural history (Dalle Carbonare L., Blood 2015). In SCD mice, FD prevented rH/R-induced bone loss compared to animals exposed to SD by decreasing osteoclast and increasing osteoblast activities. Up-regulation of molecular osteogenic markers such as Runx2 and Sparc and down-regulation of Rank and RankL, molecular markers of osteoclast recruitment and activity were observed in FD SCD mice exposed to rH/R compared to SD SCD animals. Similar changes, but to a lesser extent, were also observed in AA control mice exposed to rH/R stress. Expression of IL-6 (Il6) and matrix-metalloproteinase-9 (Mmp9) regulators which interact with RankL on osteoclastic activity and bone resorption were studied. In SCD mice, FD markedly reduced the up-regulation of both genes compared to SD SCD animals in conjunction with down-regulation of peroxiredoxin-2 (Prx2) gene expression, an important cytoprotective and antioxidant system. We also evaluated bone adipogenesis, which is believed to be an important contributor to bone impairment in SBD. Bone immunohistochemistry for Peripilin-1 which coats storage lipid droplets revealed increased adipogenesis in SD SCD mice compared to either FD SCD animals or AA controls, in association with downregulation of miR205, which decreases adipogenesis and enhances osteogenic activity. Our data thus indicate that in SCD exposed to rH/R, FD (i) improves osteoblastogenesis; (ii) decreases osteoclast activity; (iii) modulates the bone inflammatory response; iv) reduces adipogenesis. These findings provide new insights on the mechanism of action of ω-3 fatty acid supplementation on the pathogenesis of SBD and strengthen the rationale for ω-3 fatty acid dietary supplementation in SCD as a complementary therapeutic intervention targeting an amplified inflammatory response and sickle cell-related bone impairment. Disclosures No relevant conflicts of interest to declare. </jats:sec

The Pyruvate Kinase Activator Mitapivat Ameliorates Anemia and Prevents Iron Overload in a Mouse Model of Hereditary Spherocytosis

Hereditary spherocytosis (HS) is the most common cause of inherited red cell membranopathy, due to mutations in genes encoding for membrane or cytoskeletal proteins, including band 3, ankyrin, spectrin, band 4.1 or band 4.2. Membrane instability results in membrane surface area loss and generation of spherocytic red cells with elevated MCHC, decreased cellular deformability and reduced red cell survival, due to splenic sequestration. Clinical management of the hemolytic anemia due to HS depends on the age of the patient and the severity of anemia. Splenectomy is indicated in children with symptomatic anemia. A classic diagnostic test for HS is the incubated osmotic fragility: this test highlights the crucial role that ATP content plays in maintenance of normal RBC function including membrane stability: it is generally believed that the increased fragility of HS is the result of abnormal ATP depletion over the 24hr incubation. We explored the hypothesis that pyruvate kinase activator, mitapivat, by modulating ATP content could have potential beneficial effects for HS RBCs in band 4.2-/- mice, a well-established model of HS (Peters LL et al JCI 103: 1527, 1999). 4.2-/- mice exhibit moderate anemia which recapitulates most of the features of typical human HS without showing the profound anemization seen in other mouse models. Oral AG-348 administration to band 4.2-/- mice at dosages of 200 mg/kg/day over 6 months resulted in (i) improvement of anemia with reduced reticulocyte count (Hb 11.6±0.035 g/dL, n=17 vs 13.104±0.09 g/dL, n=9; P&amp;lt;0.05; retics: 11.4±0.1%, n=16 vs 7.6±0.2%, n=7 % P&amp;lt;0.05) and decrease hemolytic indices (LDH, total bilirubin) with decreased spleen weight/mouse weight ratio (8.41±0.08 vs 6.53±0.06, n=8 P&amp;lt;0.05); (ii) reduced hepatic and splenic iron overload; (iii) reduction in the proportion of phosphatidylserine positive RBCs, measured with Annexin V binding (2.4±0.02 vs 1.5±0.06 % P&amp;lt;0.05); (iv) reduction of naturally occurring antibody (NAb) bound to band 4.2-/- RBC membrane. The decreased hemolysis was associated with reduction in serum erythropoietin (EPO: 1001.2±41.7 U/L vs 472 ± 12.5 U/L, n=7, P&amp;lt;0.05), supporting the beneficial effect of mitapivat on HS anemia. These data indicate that mitapivat ameliorates anemia of band 4.2-/- mice, reduces chronic hemolysis and improves band 4.2-/- mouse RBC features. Thus, mitapivat might represent an interesting therapeutic option for HS patients. Disclosures Kung: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Kosinski:Agios Pharmaceuticals Inc: Current Employment, Current equity holder in publicly-traded company. Dang:Agios Pharmaceuticals Inc.: Current Employment, Current equity holder in publicly-traded company. Brugnara:Sysmex America Inc.: Consultancy; American Journal of Hematology: Other. </jats:sec

Increased levels of ERFE-encoding FAM132B in patients with congenital dyserythropoietic anemia type II

Recessive mutations in SEC23B gene cause congenital dyserythropoietic anemia type II (CDAII),1 a rare hereditary disorder hallmarked by ineffective erythropoiesis, iron overload, and reduced expression of hepatic hormone hepcidin.2,3 Some erythroid regulators have been proposed as pathological suppressors of hepcidin expression, such as growth differentiation factor 15 (GDF15) in thalassemia, CDAI and II,4-6 even if alone it seems not necessary for physiological hepcidin suppression.7 The most recently described is the erythroblast-derived hormone erythroferrone (ERFE), a member of TNF-\u3b1 superfamily that specifically inhibits hepcidin production. ERFE-encoding FAM132B is an erythropoietin (EPO)-responsive gene in experimental models.8 However, the function of ERFE in humans remains to be investigated. This study provides the first analysis on ERFE expression in human model of dyserythropoietic anemia with ineffective erythropoiesis, such as CDAII. Our ex vivo and in vitro data indicate that ERFE over-expression in CDAII patients might be most likely related to both physiological and pathological mechanisms leading to hepcidin suppression in condition of dyserythropoiesis. Indeed, we clearly demonstrated that in two different genetic conditions sharing common clinical findings and similar pathogenesis, such as CDAII and BT-intermedia, FAM132B over-expression is related to the abnormal erythropoiesis. Nevertheless, the absence of a clear correlation between erythroferrone levels and CDAII iron balance suggest that ERFE cannot be the only erythroid regulator of hepcidin suppression, at least in CDAII patients