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Modeling human erythroid-macrophage interactions
locytes (Online Supplementary Figure S4E,F). Flow cytome- try data showed that indeed both GC-macrophages and unstimulated cells can bind erythroid cells, however, increased cluster formation was found for GC- macrophages compared to unstimulated cells (Figure 4G). These results demonstrate that GC-macrophages func- tionally resemble specific aspects of macrophages within the erythroblastic island by binding erythroblasts and reticulocytes and phagocytosing pyrenocytes.
GC-macrophages share characteristics with CD163+ macrophages found in human BM and FL
To investigate whether GC-macrophages share pheno- typical characteristics with macrophages found in the two major erythropoietic organs during human development and adulthood (FL and BM, respectively), mononuclear cells of both organs were analyzed. Between week 15 and 22 of human development, the FL is primarily undertaking erythropoiesis, representing a median of 85% of the total number of mononuclear cells compared to 29% in BM, with increased frequencies of CD71+CD235– pro-ery- throblasts in FL (Figure 5A,B). To prevent the presence of free immunogenic pyrenocytes and to support erythroid cell requirements in the developing embryo, it is anticipat- ed that the FL contains significant amounts of erythroblas- tic islands and, thus, supporting macrophages. Indeed, Figure 5C shows a 6.5-fold increase in CD163+ FL macrophages compared to BM (3.3% vs. 0.5%). Further characterization shows only subtle differences in expres- sion of macrophage markers (Figure 5D and Online Supplementary Figure S5A,B), as both macrophage popula- tions express high levels of CD163 and CD14 and have intermediate levels of CD169, CD206 and VCAM1. CD163+ BM macrophages tend to express more CXCR4, whereas CD163+ FL macrophages have higher expression of CD16. Online Supplementary Table S3 displays the com- parison between the mean fluorescence intensity (MFI) of BM, FL, non-stimulated and GC-macrophages and reveals that GC-macrophages phenotypically recapitulate macrophages found in the FL and BM. GC-macrophages are more similar to BM macrophages (CD16 and CXCR4 expression), however, they also share features of FL macrophages (CD206 expression). Unstimulated cells do not express VCAM1, and have low expression of CD206, CD163, CD14 and CD16. Figure 5E,F shows that both BM and FL CD163+ macrophages bind erythroid cells (46% in BM vs. 83% in FL), indicating that CD163 purifies ery- throid-supporting macrophages. Interestingly, FL macrophages have increased interactions with CD71+CD235a+ cells compared to BM. The similarity of marker expression levels of BM, FL and GC-macrophages and the fact that all three populations form erythroid clus- ters suggest that GC-macrophages share phenotypic and functional characteristics with in vivo erythroid-supporting macrophages. GC-macrophages could thus be used as a substitute in vitro model to study the supportive effects of macrophages on erythropoiesis.
Discussion
We have previously shown that monocyte-derived macrophages can support erythropoiesis by increased sur- vival of HSPC.12 Herein, we show that these macrophages, derived from CD14+ monocytes, are differentiated in a glu-
cocorticoid-dependent manner (termed GC- macrophages), interact with erythroid cells of all stages and phagocytose the extruded pyrenocytes. Besides these functional aspects, GC-macrophages also share phenotyp- ic characteristics with resident macrophages from both human BM and FL, among which there is high expression of CD163 and CD206. Interestingly, CD163+ BM cells appear to be more heterogeneous compared to FL cells. GC-macrophages also phenotypically resemble macrophages described recently by Belay et al., who employed a lentivirally introduced small molecule respon- sive Mpl-based cell growth switch that enabled cord blood or BM CD34+ cells to be differentiated to erythroid- supporting macrophages.13 Similar to GC-macrophages, these cells express CD14, CD163, CD169, CD206, VCAM1, ITGAM and ITGAX. Herein, we show that these macrophages can also be differentiated from periph- eral blood monocytes using dexamethasone, without the need for genetic manipulation. Falchi et al. showed that in erythroid culture conditions, CD34+ cells can also differen- tiate to macrophages that interact with erythroid cells, however, we can exclude this differentiation pathway as the purified CD14+ monocytes we used to differentiate macrophages from peripheral blood did not show hematopoietic colony potential or CD34+ contamination.12
The erythroid system is renowned for its rapid response to systemic decreases in oxygen pressure. Together with elevated EPO levels, glucocorticoid levels also increase upon exposure to high altitude.42 EPO, SCF and glucocor- ticoids induce erythroblasts to proliferate whilst inhibiting differentiation.43-46 Elevated systemic EPO and glucocorti- coids as a response to low-oxygen stress leads to increased erythroid output due to augmented survival and prolifera- tion of BM erythroblasts. To accommodate this increased erythropoiesis, we hypothesize that the number of central macrophages must also be increased or alternatively these cells would have to engage with more erythroblasts.
Our flow cytometry and cytospin data confirmed that GC-macrophages interact with erythroid cells of all stages, be that as it may, this does not provide information on the longevity of the interactions, as these could be tran- sient, as previously implied.47 Via live cell imaging we ana- lyzed the interaction between GC-macrophages and ery- throblasts, which revealed that GC-macrophages are more mobile compared to cells that were cultured in the absence of dexamethasone, and that this mobility, or “macrophage ranging”, results in more interactions with erythroblasts. Higher mobility was accompanied by an increased expression of proteins involved in migration and motility. High motility has previously been observed in CD34+ differentiated macrophages stimulated with dex- amethasone.47 Motility is an important functional aspect, as erythroblastic islands in vivo form away from sinusoids and migrate to the sinusoidal endothelium to release retic- ulocytes into the circulation.48,49 Interestingly, this work also demonstrates that non-glucocorticoid-stimulated monocytes can interact with erythroblasts, as they form interactions for the same length of time (1.8 hours on aver- age) when they encounter erythroblasts. This suggests that both populations express receptors that allow engage- ment and interaction with erythroblasts, however, GC- macrophages have significantly more interactions with erythroblasts per macrophage and bind a higher number of erythroblasts. Surprisingly, GC-macrophages display low expression of VCAM1, suggesting that erythroblast
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