Assessing the Impact of Intravascular Hemolysis on Erythropoiesis in Sickle Cell Disease

Intravascular hemolysis is a hallmark of sickle cell disease (SCD). While it has been well established that the hemolysis-derived products, such as hemoglobin (Hb) and free heme, exert proinflammatory and pro-oxidative effects, contributing to the vascular and tissue damage in SCD, the effects of hemolysis on erythropoiesis have not been studied. We and others have reported that hemolysis in SCD led to upregulation of type I interferon IFNα. We further documented that the ability of Townes sickle mice to increase their erythropoietic capacity to compensate for anemia was impaired. To examine whether the impaired erythropoiesis in SCD is associated with the hemolysis-driven IFNα production and to define the underlying mechanisms, we injected mice with hemin, red cell lysate, or IFNα to mimic the hemolysis status in SCD and then examined erythropoiesis by colony-forming assay, flow cytometric analysis, and western blot. We also examined the effects of hemin and IFNα on erythropoiesis using an in vitro erythroid culture system. We found that intravascular hemolysis inhibited erythropoiesis in SCD through inhibition of erythropoietin (EPO)/erythropoietin receptor (EPOR) signaling via a heme-IFNα-CISH axis. Herein, we describe how to prepare hemin, red cell lysate, and IFNα, and present examples of in vivo and in vitro assays to assess erythropoiesis. Our methods can be applied to study changes in erythropoiesis in other diseases characterized by intravascular hemolysis.