EPO-Dependent Recovery of Late-Stage Erythroid Progenitors in the Marrow Precedes Splenic Expansion: Insights From a Sublethal Radiation Model

Abstract 180 Erythropoiesis is a robust process of cellular expansion and maturation that occurs in the bone marrow and spleen of mice. Following clastogenic injury such as total body irradiation (TBI), erythroblasts are severely depleted in these organs, resulting in loss of reticulocyte output and the development of a mild anemia (Peslak et al., Exp. Hematol. 2011). However, the mechanistic and microenvironmental factors underlying erythroid recovery following sublethal TBI are poorly understood. To this end, we utilized colony assays to quantify erythroid progenitors, which consist of immature d7 erythroid burst-forming units (BFU-E) and more mature d3 BFU-E and erythroid colony forming units (CFU-E). Imaging flow cytometry was used to quantify erythroblast precursors. We found that d7 BFU-E undergo a slow, incomplete recovery during the first 10 days post-4 Gy TBI of C57Bl/6 mice. In contrast, d3 BFU-E exhibit a robust recovery beginning at 4 days post-TBI that is immediately followed by a rapid increase in CFU-E numbers to over 200 percent of steady-state levels. This initial erythroid progenitor recovery is followed by a wave of erythroid precursor maturation and red cell formation that occurs in close association with macrophages in the bone marrow. These erythroblast islands undergo a rapid synchronous expansion that peaks at 6 days post-TBI, suggesting that the bone marrow microenvironment plays a role in the recovery of the erythron from sublethal TBI. We hypothesized that erythropoietin (EPO), the primary regulator of erythroid survival and proliferation, mediates the rapid, specific expansion of late-stage erythroid progenitors following radiation injury. We found that plasma EPO levels increase 13-fold 4 days after 4 Gy TBI, temporally correlated with expansion of d3 BFU-E. Furthermore, maintenance of steady-state hematocrit levels following TBI prevented EPO induction and blocked expansion of late-stage erythroid progenitors, while exogenous EPO administered at 1 hour post-radiation specifically advanced recovery of late-stage progenitors. These data indicate that EPO is required for expansion of d3 BFU-E and CFU-E following radiation-induced marrow depletion. During times of acute hypoxia, such as the severe anemia induced by bleeding or phenylhydrazine exposure, EPO production is rapidly upregulated and splenic stress erythropoiesis is induced. Surprisingly, splenic erythropoiesis is absent during the rapid initial recovery of erythropoiesis in the bone marrow at 4–6 days post-TBI. However, a massive expansion of CFU-E begins at 7–8 days post-4 Gy TBI in spleen. EPO administration at 4 days following 4 Gy TBI significantly enhances late-stage progenitor recovery exclusively in the marrow, indicating that erythroid progenitors are not present in spleen at the time of rapid bone marrow expansion and that late-stage erythroid progenitor recovery initiates in the marrow and subsequently proceeds to the spleen. Furthermore, we found that erythroid progenitors transiently emerge in the bloodstream at 6–8 days post-TBI, following marrow recovery and prior to initiation of splenic erythropoiesis. These data are consistent with endogenous migration of the erythron from the bone marrow to the spleen during recovery from radiation-induced erythroid injury. Taken together, our data indicate that recovery from sublethal irradiation injury is regulated primarily by the EPO-induced expansion of late-stage erythroid progenitors in the bone marrow. This form of clastogenic injury is critically different from bleeding or hemolysis, which preserve bone marrow and splenic erythroblasts and induce expansion of splenic erythroid stress progenitors. Sublethal irradiation injury thus provides a unique model for the in vivo study of endogenous erythroid recovery. This model may be clinically useful for the functional evaluation of therapeutic factors that regulate or modulate erythroid cell maturation. Disclosures: Bemis: Litron Laboratories: Employment, Patents & Royalties.