AbstractErythropoiesis is one of the most efficient cellular processes in the human body producing approximately 2.5 million red blood cells every second. In humans, this process occurs in the bone marrow on a highly specialised niche called the erythroblastic island. This niche is comprised of a central macrophage surrounded by developing erythroblasts. The central macrophage is known to play an important role in erythropoiesis, but it is unknown how the differentiating erythroblast is initially attracted to the macrophage to form an island. The Eph receptor family is involved in contact inhibition of locomotion which controls how cells react when they initially encounter other cells, leading to adhesion or repulsion. We set out to explore the importance of the Eph receptor family in erythroblastic island formation.
The first objective of this project was to create an assay and imaging pipeline to study the initial contact between macrophages and erythroblasts. Image analysis demonstrated we can observe long-lasting interactions as the macrophages bind one or more erythroblasts. Once this was sucessfully completed, this methodology was used to dissect the contribution different receptors made at the surface of the two cell types to island formation. We confirmed that human VCAM1+ and VCAM- bone marrow macrophages, and in vitro cultured macrophages are ephrin-B2 positive, whereas differentiating human erythroblasts express EphB4, EphB6 and EphA4. Furthermore, we detected a rise in integrin activation on erythroblasts at the stage at which the cells would bind, which is independent of Eph receptor presence. The application of a specific active integrin inhibitory peptide disrupted island formation illustrating the known importance of integrins. We next showed that specific inhibitory peptides or shRNA depletion of EphB4 also cause a significant reduction in the ability of macrophages to interact with erythroblasts. Bone marrow macrophages were also affected by EphB6 depletion illustrating a subtle difference between macrophage type. This study demonstrates for the first time that EphB expression on erythroblasts, independently from integrins, facilitates the initial recognition and subsequent interaction with macrophages.
To conclude, we have developed methodology and imaging analysis which enables us to dissect the different roles surface receptors might play within erythroblastic islands. We have shown that EphB receptors and active integrin β1 are important for the recognition of the two cell types as binding partners, and their presence ensures that if one is lost, some contacts can still occur. This methodology can be used to decorticate the different pathways involved in the formation and function of erythroblastic islands in humans. This understanding could lead to a better yield and quality of erythropoiesis conducted in vitro.
|Date of Award||23 Jan 2019|
|Supervisor||Ash M Toye (Supervisor)|