An unexpected role for maea in preventing ferroptosis of erythroblastic island macrophages to support erythropoiesis

Abstract The erythroblastic island (EBI), a functional unit consisting of a central macrophage surrounded by developing erythroid cells, was first identified as the erythropoietic niche several decades ago. EBI formation depends on direct cell-cell interactions mediated by specific adhesion molecules between macrophages and erythroblasts. Among these, macrophage erythroblasts attacher (Maea) is the first identified cell attachment molecule, expressed by both erythroblasts and central macrophages. A previous study revealed that selective deletion of Maea in macrophages, but not in erythroblasts, disrupted EBI maintenance and led to severe defects in macrophages in the bone marrow. However, the underlying molecular mechanisms by which Maea regulates the maintenance of macrophages to support erythropoiesis remain unclear. In the present study, we generated Maeafl/flmice and crossed them with EpoR-tdTomato-Cre mice which have been shown to efficiently recombine in macrophages in addition to erythroid cells. We show that Maea was deleted in EBI macrophages from the Maeafl/flEpoR-tdTomatoCre mice. The Maeafl/flEpoR-tdTomatoCre mice exhibited mild anemia as demonstrated by decreases in RBC counts, HCT and HGB along with increases in reticulocytes, blood EPO levels and spleen erythropoiesis. Analyses of bone marrow erythropoiesis revealed that although the frequencies of BFU-E, CFU-E and erythroblasts were significantly decreased in the BM of the Maeafl/flEpoR-tdTomatoCre mice compared to control mice, the colony forming ability of the sorted BFU-E and CFU-E cells did not change, indicating that the impaired BM erythropoiesis in the Maeafl/flEpoR-tdTomatoCre mice was not due to deletion of Maea in erythroid cells. This was further confirmed by our findings that specific deletion of Maea in erythroid cells using Gypa-eGFP-Cre mice which only recombines in erythroid cells, did not result in any notable phenotypic changes, even under stress erythropoiesis conditions. In marked contrast, the numbers of EBI macrophages were decreased, and the expression of adhesion molecules CD169 and CD163 on EBI macrophages were downregulated, leading to decreased EBI numbers as well as decreases in erythroid cells surrounding each central macrophage. To further investigate the molecular mechanisms for the impaired erythropoiesis in the Maeafl/flEpoR-tdTomatoCre mice, we performed single cell RNA sequencing on the enriched EBIs from control and Maeafl/flEpoR-tdTomatoCre mice. 24 cells clusters were identified and annotated to different cell types. Among these, we found lower percentage of erythroid lineage and EBI macrophages in Maeafl/flEpoR-tdTomatoCremice compared to the control mice. Genes expression comparison showed that only few genes differentially expressed genes were noted between Maeafl/flEpoR-tdTomatoCreand control mice erythroblasts, further indicating deletion of Maea has no effects on erythroid cells. In contrast, we observed a drastic alteration in the transcriptome in Maea-deficient EBI macrophages. The expression of genes involved in supporting erythropoiesis was down-regulated in Maea-deficient EBI macrophages, including the adhesion molecules CD169 and CD163, the engulfment and digestion related molecules Mertk, Timd4, Axl, Stab2 and Dnase2a, growth factors Scf, Igf1 and Vegfb, and genes involved in iron storage, absorption and transport, such as Ftl1, Fth1, Tfr, Hmox1 and Fpn1.Interestingly, genes involved in inhibition ferroptosis, such as Gpx4, pcbp2, as well as genes involved in promoting lipid peroxidation production, such as Dpp4, Sat1, were up-regulated. These data suggested that the cells might undergo ferroptosis due to dysregulated iron handing. Consistent with this, we observed significantly higher levels of cellular ROS, lipid peroxidation, and intracellular concentration of redox-active ferrous iron (Fe2+) in Maea-deleted EBI macrophages compared to controls. Importantly, Liproxstatin-1, an ferroptosis inhibitor, partially alleviated anemia symptoms, increased the numbers of EBI macrophages and EBIs, reduced levels of ROS as well as lipid ROS in EBI macrophages in the Maeafl/flEpoR-tdTomatoCre mice. Together, our findings uncovered a previously unknown role of Maea in erythropoiesis by protecting EBI macrophages from ferroptosis.