Characterization of erythroblastic island and tissue macrophages using a novel CD169-tdTomato-Cre mouse model and...

Macrophages play diverse roles, including supporting erythropoiesis. CD169 is a specific marker for macrophages. However, recent studies showed that anti-CD169 staining detected nonmacrophages, raising the concern for using anti-CD169 antibody to correctly assign macrophages. Genetically engineered mouse models provide another means of monitoring gene expression. However, no mouse model is currently available to directly monitor CD169 expression. Erythropoiesis relies on macrophage-mediated iron recycling for iron supply. Although it is established that spleen (SP) macrophages play critical roles in erythrophagocytosis and iron recycling, whether and how other tissue macrophages are involved remain incompletely understood. To address these issues, we generated CD169-tdTomato-Cre mice. We also compared various tissue macrophages by single-cell RNA sequencing, flow cytometry, and cytospin analyses. We showed that CD169-tdTomato was detected and that CD169-Cre-mediated recombination occurred only in macrophages. Notably, bone marrow (BM) F4/80⁺CD169⁺ macrophages resemble erythroblastic island macrophages, as demonstrated by the expression of Epor along with other macrophage feature genes. A comparison of tissue macrophages revealed significant differences, including erythrophagocytosis by SP and BM macrophages, but not liver macrophages, at steady state; differential expression of the macrophage surface markers CD169, Vcam1, Cx3cr1, and Csf1r and the iron recycling genes Hmox1, Spic, and CD91; and selective expression of globin genes in lung macrophages. Our findings have uncovered previously unknown differences among tissue macrophages and provide novel insights into the mechanisms by which tissue macrophages participate in iron recycling to support erythropoiesis. The novel dual-functional CD169-tdTomato-Cre mouse line will have broad applications for studies of gene function in macrophages and their role in modulating erythropoiesis.