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E. Heideveld et al.
saponin PSAP and GLB1, in addition to other lysosome/hydrolase activity-related GO-categories (Figure 2D,E). In addition, GO-categories associated with lipid metabolic processes were also downregulated in GC- macrophages. Furthermore, CHI3L1 and CD44 are highly upregulated in non-glucocorticoid stimulated cells (Figure 2B). CHI3L1 is described as a pro-inflammatory factor,29,30 while CD44 has been expressed on pro-inflammatory tis- sue macrophages.31 In conclusion, CD14+ monocytes that have been differentiated in the presence of dexametha- sone display a distinct anti-inflammatory proteomic pro- file and are further denoted as GC-macrophages, while unstimulated cells have a more inflammatory profile.
GC-macrophages are motile and bind erythroblasts
GC-macrophages may, besides supporting the erythroid yield, also regulate terminal differentiation of erythrob- lasts, recapitulating aspects of erythroblastic islands. In mice, it has been shown that BM central macrophages can bind erythroblasts through various interactions: VCAM1- integrin-α4β1,16,32 integrin-α5β1-ICAM4,33,34 erythroblast macrophage protein (EMP)-EMP,4,35 or EphrinB2- EphrinB4.36 Flow cytometry data revealed that GC- macrophages express common cell adhesion molecules (CAM), such as integrins (α4 [ITGA4], β1,2 [ITGB1, ITGB2/CD18] and αL,M,X [ITGAL/CD11a, ITGAM/CD11b, ITGAX/CD11c]), the immunoglobulin (Ig) superfamily (ICAM1, PECAM, VCAM1) and E- and L- selectin (Figure 3A and Online Supplementary Figure S3A). Most of these CAM could be identified in the proteomics data, including ICAM3, integrin-β5 and α5, however, VCAM1, selectins and EMP were not detected (Online Supplementary Table S1). With the exception of integrin-β5, these CAM were not differentially expressed between GC-macrophages and non-glucocorticoid stimulated cells. Erythroblasts expressed similar ITGA4 levels compared to GC-macrophages, but exhibited a 10-fold reduction in ITGB1 expression and low expression of ICAM1 and PECAM, whereas VCAM1 was not detected (Figure 3A). When differentiating erythroblasts towards reticulocytes (Online Supplementary Figure S3B,C), the expression of CAM was reduced, as expected, which potentially indi- cates a lower binding affinity of erythroid cells to macrophages during erythroid differentiation. Next, we investigated whether GC-macrophages interact in vitro with erythroid cells compared to non-glucocorticoid stim- ulated monocytes. Indeed, live imaging cells for 2.5 days showed that GC-macrophages are highly motile and non- stimulated macrophages are non-motile (Figure 3B), a finding which corroborates the increased expression of cell migration and motility proteins (Figure 2D) whilst engaging twice as many erythroblasts (0.5 vs. 0.3, P<0.0001) at every time point measured (Figure 3C,D). In addition, cytospins of macrophages co-cultured for 24 hours with erythroblasts showed that the number of macrophages binding erythroblasts as well as the number of erythroblasts bound was increased on GC- macrophages compared to non-GC macrophages (Figure 3E and Online Supplementary Figure S3D). Nonetheless, no difference in interaction duration between erythroblasts and macrophages from both conditions was observed (Online Supplementary Figure S3E), suggesting that the unstimulated cells possess some machinery to interact with erythroblasts. In conclusion, GC-macrophages are motile, express a variety of CAM and form erythroblast
interactions with increased frequency and numbers per macrophage compared to cells cultured in the absence of dexamethasone.
GC-macrophages express TAM-receptor family members and phagocytose pyrenocytes
As CD169+CD163+ macrophages promote erythro- poiesis,8 we decided to examine whether GC-macrophages can provide a similar functional role in vitro. In mice, pyrenocytes are phagocytosed by central macrophages in a Mer tyrosine kinase (MERTK)-depen- dent manner.18 RT-PCR showed that GC-macrophages upregulate both MERTK and AXL mRNA compared to freshly isolated and non-glucocorticoid stimulated mono- cytes (Figure 4A). MERTK expression was inhibited by mifepristone treatment during the first three days of cul- ture, whereas AXL was not, suggesting that AXL expres- sion is induced via a trans-regulated process while MERTK needs the transcriptional activity of the glucocorticoid receptor. Note that TYRO3 levels are dexamethasone- independently increased. Besides TAM-receptors, other PS-receptors on macrophages have been reported to be involved in clearing apoptotic bodies, such as TIM337 (T-cell Ig and mucin-domain containing-3), STAB38 and CD300A39 (CMRF35-like molecule 8). TIM3 mRNA levels were increased, albeit independently of dexamethasone (Online Supplementary Figure S4A). This was confirmed by mass spectrometry, as peptides corresponding to TIM3 were identified in GC-macrophages (HAVCR2 in Online Supplementary Table S1). CD300A and STAB1 were also identified, of which STAB1 was significantly increased in GC-macrophages compared to unstimulated cells. Interestingly, proteomics data showed that lactadherin, a PS-binding glycoprotein which stimulates phagocytosis of red blood cells by macrophages,40 was significantly induced in GC-macrophages compared to unstimulated cells. RT-PCR confirmed increased lactadherin mRNA lev- els, but this was dexamethasone-independent (Online Supplementary Figure S4B). Moreover, both GC- macrophages and unstimulated cells express DNASE2, a crucial protein required to degrade DNA within phagocy- tosed apoptotic bodies or pyrenocytes in macrophages.41
Expression of TAM-receptors and other PS-receptors on GC-macrophages may be a prerequisite to phagocytose particles, cells or pyrenocytes in case of erythropoiesis. Figure 4B shows that the number of GC-macrophages that phagocytose particles, in addition to the amount of zymosan particles per macrophage, is higher (73% vs. 45%, 2.3 vs. 1.7, respectively) compared to unstimulated cells. Subsequently, both unstimulated cells and GC- macrophages were co-cultured with a mixture of differen- tiating erythroblasts, reticulocytes and pyrenocytes (Online Supplementary Figure S3B,C) for 24 hours. Cytospin analysis showed that both GC-macrophages and unstim- ulated cells bind erythroid cells (Figure 4C), however, increased numbers of nucleated cells, reticulocytes and pyrenocytes bind to GC-macrophages compared to unstimulated cells (Figure 4D-F and Online Supplementary Figure S4C). Note that all nucleated erythroid cells are specifically aligned with their nucleus towards the macrophage as observed in vivo (Figure 4C). Pyrenocytes, however, were almost solely phagocytosed by GC- macrophages (Figure 4F and Online Supplementary Figure S4D). Importantly, GC-macrophages and unstimulated cells did not overtly phagocytose nucleated cells or reticu-
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haematologica | 2018; 103(3)