HMGB1-mediated restriction of EPO signaling contributes to anemia of inflammation.

Anemia of inflammation, also known as anemia of chronic disease, is refractory to erythropoietin (EPO) treatment, but the mechanisms underlying the EPO refractory state are unclear. Here, we demonstrate that high mobility group box-1 protein (HMGB1), a damage-associated molecular pattern molecule recently implicated in anemia development during sepsis, leads to reduced expansion and increased death of EPO-sensitive erythroid precursors in human models of erythropoiesis. HMGB1 significantly attenuates EPO-mediated phosphorylation of the Janus kinase 2/STAT5 and mTOR signaling pathways. Genetic ablation of receptor for advanced glycation end products, the only known HMGB1 receptor expressed by erythroid precursors, does not rescue the deleterious effects of HMGB1 on EPO signaling, either in human or murine precursors. Furthermore, surface plasmon resonance studies highlight the ability of HMGB1 to interfere with the binding between EPO and the EPOR. Administration of a monoclonal anti-HMGB1 antibody after sepsis onset in mice partially restores EPO signaling in vivo. Thus, HMGB1-mediated restriction of EPO signaling contributes to the chronic phase of anemia of inflammation.

Rasa3 regulates stage-specific cell cycle progression in murine erythropoiesis.

Inherited bone marrow failure syndromes (IBMFS) are heterogeneous disorders characterized by dysregulated hematopoiesis in various lineages, developmental anomalies, and predisposition to malignancy. The scat (severe combined anemia and thrombocytopenia) mouse model is a model of IBMFS with a phenotype of pancytopenia cycling through crises and remission. Scat carries an autosomal recessive missense mutation in Rasa3 that results in RASA3 mislocalization and loss of function. RASA3 functions as a Ras-GTPase activating protein (GAP), and its loss of function in scat results in increased erythroid RAS activity and reactive oxygen species (ROS) and altered erythroid cell cycle progression, culminating in delayed terminal erythroid differentiation. Here we sought to further resolve the erythroid cell cycle defect in scat through ex vivo flow cytometric analyses. These studies revealed a specific G0/G1 accumulation in scat bone marrow (BM) polychromatophilic erythroblasts and scat BM Ter119-/c-KIT+/CD71lo/med progenitors, with no changes evident in equivalent scat spleen populations. Systematic analyses of RNAseq data from megakaryocyte-erythroid progenitors (MEPs) in scat crisis vs. scat partial remission reveal altered expression of genes involved in the G1-S checkpoint. Together, these data indicate a precise, biphasic role for RASA3 in regulating the cell cycle during erythropoiesis with relevance to hematopoietic disease progression.

Differential effects of RASA3 mutations on hematopoiesis are profoundly influenced by genetic background and molecular variant.

Studies of the severely pancytopenic scat mouse model first demonstrated the crucial role of RASA3, a dual RAS and RAP GTPase activating protein (GAP), in hematopoiesis. RASA3 is required for survival in utero; germline deletion is lethal at E12.5-13.5 due to severe hemorrhage. Here, conditional deletion in hematopoietic stem and progenitor cells (HSPCs) using Vav-iCre recapitulates the null phenotype demonstrating that RASA3 is required at the stem and progenitor level to maintain blood vessel development and integrity and effective blood production. In adults, bone marrow blood cell production and spleen stress erythropoiesis are suppressed significantly upon induction of RASA3 deficiency, leading to pancytopenia and death within two weeks. Notably, RASA3 missense mutations in two mouse models, scat (G125V) and hlb381 (H794L), show dramatically different hematopoietic consequences specific to both genetic background and molecular variant. The mutation effect is mediated at least in part by differential effects on RAS and RAP activation. In addition, we show that the role of RASA3 is conserved during human terminal erythropoiesis, highlighting a potential function for the RASA3-RAS axis in disordered erythropoiesis in humans. Finally, global transcriptomic studies in scat suggest potential targets to ameliorate disease progression.

Increased Reactive Oxygen Species and Cell Cycle Defects Contribute to Anemia in the RASA3 Mutant Mouse Model scat.

RASA3 is a Ras GTPase activating protein that plays a critical role in blood formation. The autosomal recessive mouse model scat (severe combined anemia and thrombocytopenia) carries a missense mutation in Rasa3. Homozygotes present with a phenotype characteristic of bone marrow failure that is accompanied by alternating episodes of crisis and remission. The mechanism leading to impaired erythropoiesis and peripheral cell destruction as evidenced by membrane fragmentation in scat is unclear, although we previously reported that the mislocalization of RASA3 to the cytosol of reticulocytes and mature red cells plays a role in the disease. In this study, we further characterized the bone marrow failure in scat and found that RASA3 plays a central role in cell cycle progression and maintenance of reactive oxygen species (ROS) levels during terminal erythroid differentiation, without inducing apoptosis of the precursors. In scat mice undergoing crises, there is a consistent pattern of an increased proportion of cells in the G0/G1 phase at the basophilic and polychromatophilic stages of erythroid differentiation, suggesting that RASA3 is involved in the G1 checkpoint. However, this increase in G1 is transient, and either resolves or becomes indiscernible by the orthochromatic stage. In addition, while ROS levels are normal early in erythropoiesis, there is accumulation of superoxide levels at the reticulocyte stage (DHE increased 40% in scat; p = 0.02) even though mitochondria, a potential source for ROS, are eliminated normally. Surprisingly, apoptosis is significantly decreased in the scat bone marrow at the proerythroblastic (15.3%; p = 0.004), polychromatophilic (8.5%; p = 0.01), and orthochromatic (4.2%; p = 0.02) stages. Together, these data indicate that ROS accumulation at the reticulocyte stage, without apoptosis, contributes to the membrane fragmentation observed in scat. Finally, the cell cycle defect and increased levels of ROS suggest that scat is a model of bone marrow failure with characteristics of aplastic anemia.