Page 73 - Haematologica April 2020

EPHB4 drives macrophage-erythroblasts interactions
the formation and maintenance of human erythroblastic islands or how these two different cell types specifically recognize one another as binding partners.
The EPH receptor family is the largest tyrosine kinase receptor family.12 It is separated into two protein branches which are largely distinct: the A family and the B family.13 EPH receptors are very versatile as they can control adhe- sion, migration and proliferation;12,14,15 leading to their important role in development, in particular, through their role in contact inhibition of locomotion (CIL). One current model for CIL suggests that depending on which EPH receptors and their ligands ephrins are present and their abundance at the surface will dictate the response of cells as they come into contact.16 As both EPHB and EPHA receptors can be simultaneously expressed on the surface of cells, it is thought that the ratio of EPHA to EPHB recep- tor abundance at the surface of the cells determines the behavior of the two cells as they collide.16,17 Hence, when EPHA receptors are in excess and engage the ligand, the cells will be repulsed, whereas if EPHB are in excess and activated, this can lead to attraction and possibly drive adhesion.
Recently, several reports have discussed the importance of EPH receptor function within the BM niche. In mice, one EPHB4 ligand, ephrin-B2 is expressed on HSC and is important for the release of the progenitor cells into the bloodstream.14,18 EPHB4 is also reported to exert control over niche size, as transgenic mice that over-express EPHB4 produce more HSC cells and display a higher BM reconstitution capacity.19,20 However, the role that EPH receptors play specifically in the erythroid lineage is based primarily upon the demonstration of EPHB4 expression on human BM CD34+ cells and from the observed increase in CFU-E formation upon co-culture with stromal cells over-expressing ephrin-B2 or HSC overexpressing EPHB4.21-23 More recently, Anselmo et al.9 proposed a role for EPHB1 in the activation of integrins via an agrin-depen- dent pathway in mice and hypothesized that this facili- tates erythroblast binding to macrophages. Whether this observation extends to a human macrophage island con- text is unknown.
We find that for humans, EPHB4, EPHB6 and EPHA4 are the only EPH receptors present on erythroblasts and that these proteins are differentially expressed on the surface during terminal differentiation. Specifically, we found high EPHB4 and EPHB6 expression in the early stages of erythropoiesis, and by the reticulocyte stage, only EPHA4 is detected. We also demonstrate that during the expan- sion phase where EPHB4 and EPHB6 are highly expressed, erythroblasts also have an increase in active integrin. Using live cell imaging we show that the inhibition of EPHB4 interaction with ephrin-B2 results in a decrease in the association between erythroblasts and macrophages despite the continued presence of active integrins. This work demonstrates for the first time that ephrin/EPH interactions, as well as the presence of integrins, drive the recognition between macrophages and erythroblasts dur- ing human erythroblastic island formation.
Methods
Bone marrow isolation
Bone marrow aspirate samples were provided by Dr Michael Whitehouse (University of Bristol) with informed consent for
research use. The use of donated BM was approved by the Bristol Research Ethics Committee (REC no. 12/SW/0199). Cells were washed from a universal sample tube using HBSS (Sigma-Aldrich, Gillingham, UK) containing 0.6% acid citrate dextrose (ACD) to remove the heparin-coated beads (included to prevent coagula- tion). The red pulp was macerated onto a 70 μm filter. Cells were washed once more with HBSS and ACD, and centrifuged at 300g between washes. Red cells were lysed using red cell lysis buffer (155mM NH4Cl, 0.137mM EDTA, 1mM KHCO3, pH 7.5) for ten minutes on ice, cells were washed a further time in HBSS with ACD, counted and stored until required.
FACS
Mononuclear cells (MNC) were thawed and washed twice with PBS containing 1% BSA and 2% glucose (PBSAG). CD14+ isola- tion was performed on thawed BM MNC according to the manu- facturer's instructions (Miltenyi Biotech, Woking, UK). Cells were counted and resuspended in PBSAG and CD14-Pacific Blue, CD106-PE and CD169-APC antibodies were added for 30 minutes at 4°C in the dark. The cells were washed twice in PBSAG, and then the CD14+CD106+and CD14+CD106- populations were sort- ed using a BD Influx Cell Sorter.
Live cell imaging
Macrophages were grown for seven days, as described above, in a 24-well plate (Corning, New York, USA). At day 7, cells were labeled with Cell Tracker Green CFMDA (ThermoFisher, Loughborough, UK) following the manufacturer’s instructions. For BM macrophages, the labeling was conducted immediately after sorting. Erythroblasts on day 6 of expansion were added to the macrophages and left overnight in culture media [IMDM (Life Technologies, Paisley, UK), 3%v/v Human serum (Sigma-Aldrich), 10 U/mL erythropoietin (Roche, Basel, Switzerland) and 1mg/mL holotransferrin (Sanquin Blood Supply, Netherlands)]. The biotinylated TNYLFSPNGPIARAWGSGSK-Biotin (TNYL), EILD- VPSTGSGSK-Biotin (EIL) and the control peptide DYPS- MAMYSPSVGSGSK-Biotin (DYP) were synthesized by Cambridge Peptides UK (Birmingham, UK) and added where indi- cated. The media was changed the next day with phenol-red free imaging media with replenished peptides where indicated. The optimal final ratio of cells used was 2 erythroblasts to 1 macrophage to prevent overcrowding. The plate was imaged using the Incucyte (Essen BioScience, Welwyn Garden City, UK) every hour at 20x magnification. The spatial relationship between erythroblasts and macrophages was characterized using Fiji.24,25 Initially, lateral drift in the phase-contrast and fluorescence images over time was corrected using the StackReg plugin.26 A difference of Gaussian filter (approximating the equivalent Laplacian of Gaussian27) was then applied to the phase-contrast channel to enhance features with diameters matching those expected for ery- throblasts. Erythroblasts were subsequently identified with the TrackMate plugin using the Laplacian of Gaussian feature detec- tor.28 Fluorescence channel images were processed with rolling- ball and Gaussian filters to remove inhomogeneity of illumination and high-frequency noise, respectively. The images were then thresholded using the Otsu method29 with a user-defined fixed multiplier offset and passed through the ImageJ particle analyzer to identify macrophages. Macrophages were tracked between frames using the Apache HBase (v1.3.1; Apache Software Foundation, https://hbase.apache.org) implementation of the Munkres algorithm with costs assigned based on object centroid separation.30 Instances where objects in the phase-contrast chan- nel coincided with macrophages identified in the fluorescence channel were removed, as these likely corresponded to accidental detection of macrophages. Finally, spatial relationships between
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