Lack of Beta-1 Integrin Limits Stress Erythropoiesis and Splenomegaly in Beta-Thalassemia

After blood loss, the production of red cells must be increased by stress erythropoiesis. This phenomenon is associated with increased proliferation and reduced differentiation of the erythroblasts, leading to a net increase in the number of progenitor erythroid cells and red cells (erythron). In normal conditions, after expansion of the pool of erythroblasts, these cells eventually differentiate to erythrocytes and the anemia resolves. However, in diseases such as β−thalassemia, production of healthy mature erythrocytes is impaired, resulting in anemia. Over time, the expansion, rather than the differentiation, of the erythron further exacerbates the ineffective erythropoiesis (IE), reducing the ability of the erythroid progenitors to generate erythrocytes. Interrupting the interaction between macrophages and erythroblasts (MEI) in thalassemia models is efficacious in reducing IE and alleviating the disease phenotype. We speculate that these molecules are also responsible for the homing of erythroid progenitor cells to extramedullary organs, such as the spleen and liver. Our studies in erythroblasts indicate that integrin beta−1 (Itgβ₁) and also intracellular molecules such as focal adhesion kinase (Fak₁), Talin−1 and Sharpin might play a role in stress erythropoiesis. Furthermore, there is increased interaction between Itgb₁ and Fak₁ in erythroblasts co−cultured with macrophages as demonstrated by immunocytochemistry and in vitro proximity ligation assays. In addition, targeting either Itgβ₁ or Fak₁ prevents expansion of erythroid cells when cultured in the presence of macrophages. Strikingly, using Itgβ₁ together with Ter119 as selection parameters in flow cytometry, a distinct subset of erythroblasts, not discernable using CD44 or CD71, was observable, which we found to be part of the mixed orthochromatic erythroblast/reticulocyte population as determined with CD44 expression. Enucleation of erythroblasts was accompanied by a marked loss of Itgβ₁ expression, indicating that Itgβ₁ may be involved in erythroblast enucleation and differentiation. We crossed Hbb^th3/+ mice with animals in which Itgβ₁ or Fak₁ were floxed and carrying an inducible Cre−recombinase (Mx₁−Cre). From these animals, we investigated three different models; two obtained from breeding (Hbb^th3/+−Itgβ₁^fl/fl−Mx₁−Cre and Hbb^th3/+−Fak₁^fl/fl−Mx₁−Cre) and one by bone marrow transplant (BMT) of hematopoietic stem cells (HSCs) of Hbb^th3/+−Itgβ₁^fl/fl−Mx₁−Cre animals into wt mice to generate thalassemic animals that expressed the floxed Itgβ₁ only in hematopoietic cells. After serial administration of Poly(I)−Poly(C) [poly(I:C)] the animals were analyzed for their erythropoiesis in the bone marrow and spleen. All the animals treated with poly(I:C) showed populations of Itgβ₁ or Fak₁ negative cells in the bone marrow and spleen. This indicated that all the HSCs were successfully depleted of the Itgβ₁ or Fak₁ gene. Interestingly, the spleen weight of all the treated animals was reduced, on average, 50% compared to untreated thalassemic mice. Similar results were seen also in Hbb^th3/+−Itgβ₁^fl/fl−Mx₁−Cre animals generated through BMT. Therefore, Itgβ₁ and Fak₁ might contribute to the pathophysiology of thalassemia and their removal might result in reduced stress erythropoiesis, erythroid proliferation and, as a consequence, amelioration of splenomegaly. Iron analysis and quantification of Erythroferrone (ERFE) are in progress to evaluate the impact of depleting Itgβ1 and Fak1 on these mechanisms. We are now in the process of identifying compounds that target MEI and, in particular, Itgβ₁. Such molecules might be utilized for development of new treatments for thalassemia or additional disorders of aberrant erythropoiesis.