Macrophages Regulate Stress Erythropoiesis Through Direct Cellular Interactions Associated With Integrin β1-Focal Adhesion Kinase Signaling
Authors: Crielaard Bart J , Casu Carla , Ramos Pedro , Gardenghi Sara , Breda Laura , Gupta Ritama , Rivella Stefano 
Published: October 2019

Abstract
Macrophages, strategically positioned in the center of erythroblastic islands while surrounded by developing erythroblasts (EB), are important for both steady state and stress erythropoiesis. In addition to their function in iron recycling and disposal of nuclei expelled by the maturing red cells during differentiation, macrophages are able to drive erythropoietic activity directly, making them, along with erythropoietin and iron, key regulators of erythropoiesis. This stress erythropoiesis-supporting macrophage activity (SEMA) has been demonstrated recently; there is, however, only limited understanding with regard to the exact cellular mechanism by which the macrophage activity is conveyed to the proliferating erythroid cells.
Erythroblastic signaling through integrin β1 (Itgb1) and, further downstream, focal adhesion kinase-1 (Fak1), has previously been linked to stress erythropoiesis. Therefore, the current study explored the role of the Itgb1/Fak1 pathway in the macrophage-EB interplay, as well as its therapeutic potential in disorders marked by chronic stress erythropoiesis (CSE).
Mice with beta-thalassemia intermedia (Hbbth3/+ or BTI), which present with ineffective erythropoiesis, characterized by high proliferation but limited differentiation of (CD71+) erythroblasts, anemia, and splenomegaly, were used as a model of CSE. In BTI mice, the depletion of macrophages by intravenous administration of clodronate-loaded liposomes (Clod) resulted in an augmentation of thalassemic erythroid maturation, characteristically marked by a decrease of immature (CD71+Ter119+) and increase of mature (CD71-Ter119+) erythroid cells in bone marrow; a reduction in actively cycling erythroblasts in the spleen, i.e. percentage of cells in S-phase (Clod (20%) compared to PBS (40%) PBS, p<0.01); an attenuation of anemia (Hb 9 g/dL (Clod) vs 8 g/dL (PBS) p<0.01); and an amelioration of splenomegaly (weight similar to wt upon 2 month Clod (0.1 g), versus PBS-treated (0.3 g), p<0.001) and splenic architecture.
To focus on the macrophage-EB interaction in specific, human erythroid islands were generated in vitro after isolation of macrophages and CD34+ EBs from the peripheral blood of volunteers, allowing the evaluation of proliferation, expansion and differentiation of EBs upon co-culture with macrophages. Cycling and proliferation of EBs were significantly increased in the presence of autologous macrophages (60-fold increase in cell number after 8 days; versus 10-fold increase for EBs cultured alone, p<0.001), whereas erythroid enucleation, as a measure of differentiation, was reduced. Subsequent transwell co-cultures demonstrated that these effects on EBs resulted from direct cellular interactions between EB and macrophage, rather than indirect (secreted) factors.
To investigate the role of Itgb1-Fak1 signaling in the macrophage-EB crosstalk, the expression of both proteins was studied further in vitro. Flow cytometry analysis demonstrated higher surface expression of Itgb1 by EBs co-cultured with macrophages, as compared to EBs cultured alone, even though the expression of other differentiation markers was unchanged; and Fak1 expression was increased in macrophage-cultured EBs isolated from healthy volunteers, as well as from patients with beta-thalassemia major or Polycythemia Vera. Finally, a Fak1 inhibitor (FAK inhibitor 14) was used to test whether inhibition of Fak1 (Fak1i) could reverse the effect of macrophages on EB proliferation and maturation, thereby suggesting a link between erythroblastic Fak1 and the regulating effect of macrophages on erythroid development. In human CD34+ cells in vitro, Fak1i abolished the effect macrophages on the proliferation of EB, resulting in proliferation similar to that of EBs cultured alone, whereas the direct effect of Fak1i on EB proliferation was limited. In vivo, Fak1i administration to mice with BTI, rapidly reverted splenomegaly (0.2 g FAK1i vs 0.3 g PBS after 2 days, p<0.001) and ameliorated anemia (Hb 9 g/dL FAK1i vs 8.5 g/dL PBS after 2 days, p<0.05), by reducing erythroid expansion and improving EB maturation. Further analyses to identify additional partners of these proteins are underway.
In conclusion, in the present studies Itgb1 and Fak1 signaling were correlated to the mechanism by which macrophages contribute to SE and CSE, which may have critical scientific and therapeutic implications in the future.

Disclosures:
Rivella: Novartis: Consultancy; Bayer: Consultancy; Isis: Consultancy, Research Funding; Merganser: Equity Ownership, Research Funding; Biomarin: Consultancy; Alexion: Consultancy; Imago: Consultancy.