Identification And Biological Characteritic Study Of Erythroblastic Island Macrophage

Background and purposeThe erythroblastic island,composed of a central macrophage and surrounding erythroid cells,was the first hematopoietic niche discovered.The regulation of EBI macrophages during erythropoiesis is a research hotspot and difficult issue in the field of erythropoiesis.For many years,researchers have found a variety of possible surface markers of EBI macrophages.However,due to the lack of isolation of EBI macrophages,the identity of EBI macrophages per se and the regulation of erythropoiesis has thus far remained elusive.Given that EPO is essential for erythropoiesis and that Epor is expressed in numerous non-erythroid cells,we hypothesized that EBI macrophages express Epor so that EPO can act on both erythroid cells and EBI macrophages simultaneously to ensure efficient erythropoiesis.To test this notion,we employed Epor-eGFPcre knock in mouse model,human fetal liver(FL)and in vitro co-culture system to study the EBI macrophages systematically.Flow cytometry,Imagestream,RNA-seq,Single cell RNA-sequence(Sc-RNA-seq)and other methods were used to conduct the identification and isolation of EBI macrophages system to clarify the biological characteristics of EBI macrophages.The project will provide important resources for future studies of EBI macrophage function during normal as well as disordered erythropoiesis in hematologic diseases such asβ-thalassemia,PV,MDS and so on.Research methods(1)Identification,isolation and immunophenotypically analysis of EBI macrophages:To determine whether all the F4/80~+Vcam1~+CD169~+macrophages are EBI macrophages,we quantified the numbers of erythroblasts and F4/80~+Vcam1~+CD169~+macrophages in mouse BM.Then,we isolated EBI macrophages from Epor-eGFPcre mouse BM,FL and Huam FL using flowcytometry,cell sorting and Imagestream.Immunophenotypic was analyzed based on known surface molecules,which were reported to support erytghropoiesis.(2)The study of EPO on EBI formation:Then,we calculated the numbers of EBI,EBI macrophage,and erythroblast using flow cytometry,imagestream and cell counting in both mouse BM and SP after EPO injection;The expression of adhension mulecules CD163,CD169 and Vcam1 in mouse BM EBI macrophages was determined using flow cytometry after EPO injection at the same time;The expression of CD163,CD169 and EPOR were checked in human CD34~+cells derived macrophages by flow cytometry and the expression of EPOR were confirmed by EPO stimulation experiment;EBI forming assay was performed using late stage erythroblasts and human CD34~+cell-derived macrophages to test the hypothesis EPO plays a role in EBI formation by acting on EPO-responsive EBI macrophages.(3)Transcriptome analysis of EBI macrophages:RNA-seq was performed on both mouse BM EBI macropahges and human FL EBI macrophages;Bioinformatic softwares were used to analyze the data to develop a comprehensive characterization of the EBI macrophages at the molecular level in both mouse and man.q RT-PCR and Flowcytometry or Western Blot were used to confirm both m RNA and protein levels of specific-high expression molecules from RNA-seq.(4)The heterogeneity of human FL EBI macrophages:The different types of EBI were confirmed using flow dimensionality reduction analysis,cytospins and imagestream in human FL.10X genomic Sc-RNA-seq was performend for both sorted CD163~+EPOR~+and CD163~+EPOR~-macrophages.The data was analyzed using bioinformatic software to interpret different functional subpopulations of EBI macrophages.ResultsIdentification,isolation and immunophenotypically analysis of EBI macrophages(1)Calculation of the cell numbers revealed that there were～4 million F4/80~+Vcam1~+CD169~+macrophages and～10.5 million erythroblasts in mouse BM cells from two femurs and two tibias,giving a ratio of F4/80~+Vcam1~+CD169~+versus erythroblasts about 1:2.6,strongly suggesting that it is unlikely that all the F4/80~+Vcam1~+CD169~+macrophages are EBI macrophages.About 5%and 31%of F4/80~+macrophages express Epor-eGFP in mouse BM and FL,respectively.The sorted F4/80~+Epor-eGFP~+macrophages and F4/80~+Epor-eGFP~-macrophages are morphologically different in both mouse BM and FL,further demonstrating that they are indeed distinct macrophage populations.(2)Imagestream results revealed that more than 90%EBIs were formed by the F4/80~+Epor-eGFP~+macrophages in both mouse BM and FL.Further analyses showed that the percentages of EBIs with 3,4,and 5 or more erythroid cells were～20%,～26%and～54%,respectively in mouse BM.And the percentages of EBIs with 3,4,and 5 or more erythroid cells were～10%,～20%and～70%,respectively in mouse FL.Imagestream results also revealed that more than 90%of macrophages phagocytizing nuclei were F4/80~+Epor-eGFP~+macrophages in both BM and FL.Finally,our results demonstrated that Vcam1,CD169,and ER-HR3 were expressed on more than 90%of F4/80~+Epor-eGFP~+EBI macrophages,while CD163,Ly6C and CD11b were expressed on about～35%,～52%,～32%of the F4/80~+Epor-eGFP~+EBI macrophages,respectively.Additionally,Ly6G,previously implicated as an EBI macrophage marker,was not expressed on the F4/80~+Epor-eGFP~+EBI macrophages.(3)Flowcytometry results showed that all F4/80~+Epor-eGFP~+macrophages were CD45~+,almost all of them expressed Vcam1 and CD169,a proportion of them expressed CD163(～35%),Ly6C(～69%)or ER-HR3(～90%),and some of them express moderate levels of CD11b.Our results also showed that F4/80~+EporeGFP~-macrophages were CD45~+.In contrast,although almost all F4/80~+Epor-eGFP~-macrophages also expressed Vcam1 and CD169,but expression levels of Vcam1 and CD169 on the F4/80~+Epor-eGFP~-macrophages were low.Moreover,the F4/80~+Epor-eGFP~-macrophages did not express CD163,and a proportion of them expressed Ly6C(～57%),ER-HR3(～52%)and CD11b(～68%).These results indicate that mouse BM F4/80~+Epor-eGFP~+macrophages are immunophenotypically distinct from F4/80~+Epor-eGFP~-macrophages.(4)In human FL,within the CD163~+macrophages,～27%were EPOR~+.Cytospin images showed that CD163~+EPOR~+macrophages were morphologically different from CD163~+EPOR~-macrophages;Imagestream revealed that human FL native EBIs were also formed by CD163~+EPOR~+macrophages.The study of EPO on EBI formation(1)The expression of Epor in mouse BM F4/80~+Epor-eGFP~+EBI macrophages was further confirmed by the finding that EPO treatment led to phosphorylation of STAT5.EPO injection lead to an～3 folds increase in the number of EBIs with a concomitant increase in EBIs with 5 or more erythroid cells.The increase in EBIs was accompanied by an increase in both erythroblasts and F4/80~+Epor-eGFP~+macrophages,indicating the function of these Epor-eGFP~+EBI macrophages in EPO induced stress erythropoiesis.EPO injection resulted in increased surface expression of Vcam1 but not CD169 on the F4/80~+Epor-eGFP~+macrophages.Although EPO injection did not increase the surface expression of CD163,it increased the percentage of CD163 in F4/80~+Epor-eGFP~+macrophages.Importantly,stress splenic EBIs were also formed by the F4/80~+Epor-eGFP~+macrophages and the numbers of erythroblast,EBI macrophage and EBI were also significantly increased.(2)EPOR,along with CD163,and CD169 were expressed on CD34~+cell-derived macrophages.EPOR expression was further confirmed by the finding that EPO treatment led to phosphorylation of STAT5 and AKT,downstream targets of EPO/EPOR mediated signal transduction.EPO plays a significant role in EBI formation by acting on EPO-responsive EBI macrophages in vitro.Transcriptome analysis of mouse and human EBI macrophages(1)Gene Ontology(GO)analysis of the differentially expressed genes revealed that the top upregulated pathways in F4/80~+Epor-eGFP~+macrophages included erythrocyte development,receptor-mediated endocytosis,apoptotic cell clearance and cellular iron ion homeostasis,In contrast,the top upregulated pathways in F4/80~+Epor-eGFP~-macrophages were mostly related to immune and inflammatory response.Specifically,the expression levels of genes encoding proteins known to be important for EBI macrophage function in supporting erythropoiesis were significantly higher in F4/80~+Epor-eGFP~+macrophages than in F4/80~+Epor-eGFP~-macrophages.These included growth factor Igf1,Il18 and so on,adhesion molecules Vcam1,CD169 and CD163,iron metabolism molecules,phagocytosis and digestion related molecules Mertk,Timd4,Dnase2a,Trf,Homox1,Slc40a1 and so on.Additionally,four transcriptional factors(TFs)Klf1,Spic,Nr1h3 and Maf are expressed in the F4/80~+Epor-eGFP~+macrophages but not in the F4/80~+Epor-eGFP~-,suggesting their selective roles in transcriptional regulation of the EBI macrophages.(2)Gene Ontology(GO)analysis of the differentially expressed genes revealed that the top upregulated pathways in CD163~+EPOR~+macrophages included lipoprotein metabolic process,receptor-mediated endocytosis,cell adhesion and extracellular matrix orgnization.In contrast,the top upregulated pathways in CD163~+EPOR~-macrophages were mostly related to inflammatory response,immune response and chemotaxis.Specifically,the expression levels of genes encoding proteins known to be important for EBI macrophage function in supporting erythropoiesis were significantly higher in CD163~+EPOR~+macrophages than in CD163~+EPOR~-macrophages.These included growth factors IGF2 and CXCL12,adhesion molecules CD163,CD169,VCAM1,and EMP,iron metabolism molecules,phagocytosis and digestion related molecules HMOX1,SLC40A1,TIMD4,AXL,MERTK,TRF and so on.Additionally,seven TFs SPIC,NR1H3,PPARG,KLF1,TLX1,EPAS1 and ETV5 macrophages,suggesting their selective roles in transcriptional regulation of the EBI macrophages.These findings strongly suggest that the CD163~+EPOR~+macrophages in huam FL have evolved a specialized function in supporting erythropoiesis.The heterogeneity of human FL EBI macrophages(1)Cytospins and Imagestream results showed that different types of EBI existed in human FL,and the results of flow dimensionality reduction analysis showed that EBI macrophages were significantly heterogeneous.(2)The Sc RNA-seq results showed that there were four different functional subpopulations of EBI macrophage in human FL.One cluster mainly secreted growth factors to support the proliferation of early stage of erythroblasts;One cluster surrounded by different stages of erythroblasts,which surveillanced the process of erythropoiesis and cleard the abnormal erythroid cells;One cluster was considered to support the maturation of late stage of erythroblasts and one cluster was mainly correlated with digesting the erythroblast nucleus.Conclusions(1)EBI macrophages express EPOR in both mouse BM,FL and human FL,which response to EPO and then promote EBI formation.(2)EBI macrophages promote erythropoiesis by secreting growth factors,directly adhering to erythroblasts,providing iron for late stages of erythroblasts,and phagocytosising pyrenocyte.(3)EBI macrophages are heterogeneous and there are four different functional subpopulations of EBI macrophage in human fetal liver.