Macrophages Impair Erythroid Development in β-Thalassemia Intermedia
Authors: Casu Carla , Ramos Pedro , Guy Ella , van Rooijen Nico , Grady Robert W , Rivella Stefano 
Published: October 2019

Abstract
Abstract 1035
β-Thalassemia is a disorder associated with abnormal β-globin production, leading to anemia, extramedullary hematopoiesis (EMH), a decreased lifespan of the red cells and iron overload. In this disorder erythropoiesis is ineffective due to increased erythroid apoptosis and erythroblast proliferation, as well as deficient differentiation. Recent evidence suggests that erythroid development, especially under conditions of anemia (stress erythropoiesis), is highly dependent on microenvironmental factors within the erythroid niche, potentially mediated by the interaction of erythroblasts with macrophages. However, little is known about the function of these cells in pathological anemias associated with abnormal erythropoiesis. Our goal was to study the role of macrophages in normal, stress and ineffective erythropoiesis (IE).
Macrophages were eliminated by intravenous administration of clodronate-containing liposomes. Treatment was carried out for up to 12 weeks, serial measurements being made of erythropoietic and pathological parameters. As a model of stress erythropoiesis, phlebotomized wt mice were used. To study IE we utilized th3/+ mice, a model of β-thalassemia intermedia (TI).
Clodronate treatment effectively depleted splenic and bone marrow (BM) macrophages as shown by FACS and immunohistochemical analyses. Depletion of macrophages in wt mice had little effect on steady state erythropoiesis. In contrast, clodronate treatment drastically impaired the response to stress erythropoiesis in these mice, as shown by the slow recovery from phlebotomy-induced anemia. This was associated with a very slow rate of RBC and reticulocyte production, suggesting that erythroid activity was markedly impaired. Accordingly, mice depleted of macrophages were unable to expand their pool of erythroid progenitors in the BM and spleen in response to anemia, suggesting that macrophages play a critical role in this process. A similar defect was observed in response to Epo stimulation, suggesting that an intact erythroid niche is essential for normal activity of Epo in promoting erythroid expansion. Interestingly, TI mice treated with clodronate exhibited an improvement of the thalassemic phenotype. Within 40 hours of clodronate treatment, mice showed an increase in hemoglobin (Hb), RBC and reticulocyte counts in the peripheral blood, and a reduction of extra-medullary hematopoiesis (increased ratio of mature to immature erythroid cells) and splenomegaly (P<0.05 for all parameters analyzed). This indicated a more effective erythropoiesis in the absence of macrophages, suggesting that these cells negatively influence erythroid development in this disorder. Improvement of anemia was maintained for up to 12 weeks of continuous treatment, and was associated with increased RBC counts. Under these conditions, serum iron was markedly decreased, potentially reducing iron delivery to maturing RBCs. Recent studies have suggested that lowering iron delivery to erythroblasts leads to improvement of the RBC phenotype in this disorder. Consistently, MCHs were decreased after macrophage depletion, which correlated with lower accumulation of alpha-globin/heme precipitates in the RBC membranes. Moreover, the RBC lifespan in clodronate-treated mice was increased compared to that in PBS controls. This difference was maintained even when these cells were transfused into wt mice, suggesting that it was not associated exclusively with deficient RBC clearance in macrophage-depleted mice.
In conclusion, our data suggests that macrophages have two major roles in β-thalassemia: 1) to modulate iron availability for erythroid cells; 2) to impair erythroid development, as suggested by the amelioration of splenomegaly and EMH observed after clodronate treatment. We hypothesize that the macrophages within erythroblastic islands control erythropoiesis, acting as modulators of this process. Under conditions of stress erythropoiesis they positively influence erythroid development, promoting proliferation to increase the pool of erythroid cells. However, under conditions of chronic stress such as in TI, macrophages limit differentiation and promote excessive expansion of the erythron, contributing to IE.

Disclosures:
No relevant conflicts of interest to declare.