Page 44 - Haematologica3

E. Heideveld et al.
interactions may also occur in a VCAM1-independent manner. Indeed, Ulyanova et al. reported that Vcam1-/- mice do not display a compromised erythroid stress response in spleen and BM.17 Whether another interaction substitutes for VCAM1 would need to be determined. The presented monocyte differentiation methodology has potential to be exploited as an imaging platform to delin- eate the hierarchy of contributions of various receptors within the macrophage-erythroblasts in BM and GC- macrophages in future studies.
We have also demonstrated, using proteomics and imaging, that GC-macrophages actively phagocytose pyrenocytes and express the correct putative machinery to recognize pyrenocytes. The mechanism(s) through which macrophages recognize reticulocytes but phagocytose pyrenocytes are ill-defined in human erythropoiesis. Our proteomic study and RT-PCR data demonstrate that GC- macrophages express all TAM-receptors, including MERTK and other PS-receptors, which may be used by GC-macrophages to take up pyrenocytes. This work, alongside our ability to manipulate erythroblast protein expression, now provides an excellent accessible model system to mechanistically understand how macrophages promote erythropoiesis and eventually target pyrenocytes for phagocytosis and destruction. Furthermore, it is inter- esting to note that GC-macrophages interact preferably to the polarized nuclear side of erythroid cells as observed in BM erythroblastic islands. In general, proteomic analysis revealed an array of processes and proteins that are differ- entially regulated between GC-macrophages and unstim- ulated cells. The data will allow further studies to delin- eate essential pathways that are key to glucocorticoid- stimulated differentiation of monocytes towards ery- throid-supporting GC-macrophages. This is probably the concerted action of multiple pathways.
Finally, our observations have important implications for our understanding of the dynamics of the macrophage populations in human BM. We characterized both human BM and FL macrophages and found that CD163+ FL macrophages define a homogeneous population. In con- trast, CD163+ BM macrophages show a more heteroge- neous population, reflecting that CD163+ cells represent a mixed population of myeloid cells. Both human BM and FL CD163+ macrophages are capable of binding erythroid cells, however, this percentage is lower in BM (46%) com- pared to FL (83%). The FL is primarily performing erythro- poiesis at week 15-22 of embryonic development, which suggests that CD163 purifies mainly central macrophages. The reduced erythroid-macrophage clusters in BM may reflect a more heterogeneous CD163+ population with possibly different functions. Changes observed in marker expression of macrophages in both organs could thus be due to this heterogeneity in the BM population. CD163
isolation in combination with single cell RNA-sequencing may discriminate these different populations and identify specific discriminatory cell surface markers to allow for functional experiments.
Albeit for decades it was believed that all macrophages originate from monocytes,50 recent parabiosis and fate- mapping studies showed that most resident macrophages are maintained independently of monocytes.51 However, Theurl et al. showed that resident Kupffer cells in the liver contain a mixture of de novo hematopoiesis-derived and embryonic-derived macrophages. They identified an on- demand mechanism to facilitate quick and transient increases in cells that can function as Kupffer cells but orig- inate from classical monocytes.52 Taken together with our work, we hypothesize a new scenario in which specific macrophages originate from different sources depending on the need of a specific tissue or process. These processes may also occur in other tissues in response to stress, like the BM. The origin and homeostasis of human BM resi- dent macrophages is presently ill-defined, if described at all. Elevated glucocorticoid levels may lead to direct differ- entiation of monocytes and elevated numbers of nursing central macrophages to facilitate the increased erythroid output in analogy to Kupffer cells. Active research is aimed at unraveling the origin of tissue resident macrophages, which is important in order to understand not only home- ostatic but also pathogenic erythropoiesis in which a driv- ing role of macrophages has been implicated, such as poly- cythemia vera and β-thalassemia. Herein, we provide evi- dence that monocytes can indeed differentiate in vitro to macrophages that support erythropoiesis, providing a model to study such erythroid-macrophage interactions.
Funding
This work was supported by grants from the Landsteiner Foundation (LSBR1141; EA and EH and LSBR1517; MB), a NHS Blood and Transplant (NHSBT) R&D grant (WP15-05; AMT), National Institute for Health Research (NIHR) for a Blood and Transplant Research Unit in Red Blood Cell Products at the University of Bristol in partnership with NHSBT (AMT and LAH-O) and the Wellcome Trust (105385/Z/14/Z; LAH- O and ISSF; SJC). This article presents independent research partly funded by the NIHR. The views expressed are those of the authors and not necessarily the NHS, the NIHR or the Department of Health.
Acknowledgments
The authors would like to thank the staff of the CASA clinic in Leiden for collecting human fetal tissues and Dr. Tom Cupedo, Natalie Papazian and Martijn Bogaerts from the Erasmus Medical Center, Rotterdam, for providing human fetal liver mate- rial. We also thank the Central Facility of Sanquin for technical assistance regarding ImageStreamX.
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