Removal of Macrophages From the Erythroid Niche Impairs Stress Erythropoiesis but Improves Pathophysiology of Polycythemia Vera and Beta-Thalassemia
Authors: Ramos Pedro , Casu Carla , Gardenghi Sara , Breda Laura , Crielaard Bart J , Gupta Ritama , Guy Ella , Mariongiu Maria F , Levine Ross L. , Abdel-Wahab Omar , Ebert Benjamin L. , van Rooijen Nico , Ghaffari Saghi , Grady Robert W , Giardina Patricia , Rivella Stefano 
Published: October 2019

Abstract
Abstract 81
We investigated the contribution of macrophages to physiological and pathological conditions in which erythropoietic activity is enhanced. We utilized mouse models of a) anemia by phlebotomy-induced stress erythropoiesis (SE); b) increased erythropoiesis by erythropoietin (Epo) administration; c) Polycythemia Vera (Jak2V617F/+ or PV) and d) beta-thalassemia intermedia (Hbbth3/+ or BTI) in which macrophages were chemically depleted by injection of liposome-clodronate (LC).
While chronic injection (up to 3 months) of LC in normal mice had little effect on steady state erythropoiesis, depletion of macrophages severely impaired recovery from anemia following phlebotomy and significantly limited the increase in hematocrit (Htc) in animals treated with Epo. To exclude that this effect was mediated by decreased serum iron parameters, we used mice iron overloaded by dietary means or affected by hemochromatosis (Hfe-KO and Hamp-KO). In these mice, recovery from anemia was still impaired following macrophage depletion, even though serum iron and transferrin saturation levels were elevated and unaffected by LC administration.
In vitro studies using both mouse and human primary erythroblasts (EBs) indicated that EBs in S-phase were twice as many compared to EBs cultured in absence of macrophages. The numbers of terminally mature erythroid cells were up to six fold higher in co-culture conditions. Experiments using transwells indicate that direct contact between EBs and macrophages was required to generate this effect.
Since our data highlighted an important role of macrophages in enhancing erythropoiesis, we investigated two disorders in which the pool of erythroid progenitor cells is expanded, such as PV and BTI. Chronic administration of LC in PV mice completely reversed splenomegaly and the Htc (P<0.001). BTI mice exhibited normal spleen, amelioration of ineffective erythropoiesis (by accelerating the differentiation of EBs to erythrocytes), improvement of red blood cell (RBC) morphology, red cell distribution width (RDW, P<0.001) and increased hemoglobin levels (∼2g/dL, P<0.01). This effect was due to an increased RBC lifespan following LC administration (P<0.001), which was associated with a decrease in hemichrome formation, but not with a reduction in erythophagocytosis.
Our observations indicate that macrophages directly modulate stress- and pathological erythropoiesis. Several adhesion molecules participate in the formation of interactions within the erythroblastic islands, including integrins. Interestingly, βeta1integrin and its associated protein, focal adhesion kinase-1 (Fak1), were reported to be necessary for the compensatory response to anemia, suggesting that this pathway might be involved in the macrophage-EB cross-talk. More EBs co-cultured with macrophages retained cell surface expression of βeta1integrin molecule during the last stage of cell differentiation compared to EBs cultured alone, even though other differentiation markers did not shown any variation. Fak1 phosphorylation in EBs was induced by co-culturing them with splenic macrophages, suggesting that Fak1 signaling is one of the pathways activated in EBs through contact with macrophages. Administration of a FAK1 inhibitor (FAK1i) decreased proliferation of EB co-cultured with macrophages, while delayed recovery from anemia and decreased the spleen size in phlebotomized animals (40% decrease compared to phlebotomized control animals at day 4; P=0.032). Finally, short-term administration of FAK1i to BTI animals rapidly reverted splenomegaly with a concurrent reduction of erythroid expansion in both BM and spleen and led to amelioration of anemia, supported by increased RBCs count.
Our data indicate that, while macrophages allow proper erythroid response under conditions of induced anemia or increased erythropoiesis in wt mice, they contribute to the pathological progression of PV and BTI. Activation of Fak1 promotes erythroid proliferation and pathological development, while its inhibition limits ineffective erythropoiesis and splenomegaly in BTI. In conclusion, we identified a new mechanism contributing to the pathophysiology of these disorders, which we believe will have critical scientific and therapeutic implications in the near future.

Disclosures:
Levine: Agios Pharmaceuticals: Research Funding. Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding.