A Putative Erythroid Synapse Linking Developing Erythroblasts and Macrophages in Erythroblastic Islands

Abstract

An estimated 25% of the global population suffer from anemia, a major cause being deficient erythropoiesis. Mammalian erythropoiesis in the adult occurs primarily in the bone marrow (BM), with the spleen being an active site of extramedullary erythropoiesis during anemic stress. Here, we examine regulators of the anemic stress response using an unbiased transcriptomic approach to compare FACS-sorted splenic Ter-119+ erythroid precursors, from untreated mice or phenylhydrazine (PHZ)-treated mice exhibiting chemically-induced anemia. Transcriptomic analyses revealed unexpected expression of a broad range of immune markers in untreated mouse splenic erythroid cells, most which were conspicuously diminished in expression during PHZ-induced anemic stress. These surface antigens, previously unreported on erythroid cells, include markers of B lymphocytes (CD22, CD74), T lymphocytes (CD2, CD3ε) and leukocytes including CD11a/LFA-1, CD45, CD48, CD53, as well as the homing receptors L-selectin and PSGL-1. Immunophenotyping confirmed expression of these immune markers on a subpopulation of steady state BM and splenic erythroid precursors, with a significant reduction in immune marker-expressing erythroid cells seen in the spleen during anemic stress. Erythropoiesis is governed by a close relationship between erythroid precursors and central macrophages in multicellular clusters termed erythroblastic islands (EBIs). Confocal microscopy demonstrated localization of the immune markers CD2, CD3ε, CD11a and PSGL-1 on Ter-119+ mouse erythroid precursors with frequent clustering of markers seen at intercellular contact points where erythroid cells interact with the central EBI macrophage. PHZ-treated mice exhibited a significantly reduced frequency of EBIs in both the BM and spleen compared to control mice. PHZ-treated mice also demonstrated downregulated Interferon Regulatory Factor-8 (Irf8) transcripts on transcriptomic analysis of splenic erythroid precursors. Irf8-deficient mice have been reported to have a profoundly reduced erythroid compartment. There are also reports that Irf8 may be a regulator of macrophage morphology. We therefore studied Irf8-deficient mice to evaluate whether Irf8 has a role in EBI-regulation. BM analysis of Irf8-deficient mice showed a reduced frequency of EBI-like structures compared to wildtype (WT) mice. Scanning electron micrographs revealed that Irf8-/- macrophages are smaller with fewer erythroid precursors attached compared to WT macrophages. Irf8-/- EBIs harbored no identifiable reticulocytes in contrast to WT EBIs. EBI rosette reconstitution assays demonstrated that Irf8-/- erythroid precursors were unable to bind to WT or Irf8-/- macrophages, whereas Irf8-/- macrophages could interact with WT erythroid cells. Immunophenotyping of Irf8-/- erythroid precursors revealed that immune marker expression (CD2, CD38, CD43, CD45, PSGL-1) was significantly lower compared to WT counterparts. These data suggest that Irf8 may regulate both macrophage morphology and erythroid cell surface phenotype influencing erythropoiesis. The newly uncovered immune markers expressed by erythroid cells are also involved in formation of immunological synapses between lymphocytes and macrophages. This led us to question whether an analogous synapse-like structure may exist in EBIs. Serial block face scanning electron microscopy (SBF-SEM) was used to assess EBI morphology at the nanometer scale. SBF-SEM micrographs show erythroblasts extending pseudopods towards the macrophage for attachment. Three-dimensional volumetric reconstruction of SBF-SEM micrographs revealed en face spatial topologies present on the surface of EBI central macrophages similar to those seen in immunological synapses. Our findings suggest that immune markers, possibly mediated by Irf8, may function to form synaptic-like interactions between erythroid cells and macrophages that result in EBIs - an "erythroid synapse". Failure in forming these interactions may result in alterations in EBI formation with potential downstream impacts on erythropoiesis. Improved understanding of how EBIs form and function may aid in the development of therapeutic agents to combat anemia.