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Title: Investigating the support mechanisms provided by macrophages in human erythropoiesis
Author: Diez-Sevilla, Isabel
ISNI:       0000 0004 7652 1814
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2017
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Definitive erythropoiesis takes place within the bone marrow in erythroblastic islands, where the central macrophage plays a role in the expansion and terminal differentiation of surrounding erythroid cells. Central macrophages have been shown to promote erythropoiesis, although the precise mechanisms of how they regulate erythroblast development are still largely undefined. Furthermore, clinical and experimental studies suggest that macrophages treated with dexamethasone or IL10 may have an enhanced ability to support erythropoiesis. Therefore, this thesis investigates the effects of growing autologous human peripheral blood mononuclear cell-derived macrophages and erythroblasts in direct contact using an adapted in vitro culture system and the mechanisms of enhanced erythropoiesis during erythroblast co-culture with dexamethasone or IL10 treated macrophages. Supplementation of MCSF differentiated macrophages for four days with dexamethasone (10-6M) or IL10 (10ng/ml) induced M2c phenotypes (CD163+CD169+CD16+HLA-DR-) but promoted differential expression of surface markers CD14, VCAM1, CD64, CD86 and CD80. These dexamethasone or IL10 modulated macrophages substantially improved erythroblast expansion after 48 hours (1.73 ±SEM 0.39-fold; p=0.0401 and 1.68 ±SEM 0.35-fold; p=0.0397, respectively, compared to co-culture with untreated macrophages). The enhanced erythroid expansion was found to be a result of increased proliferation as opposed to heightened survival. However, erythroblast maturation, as defined by the appearance of a CD71+glycophorin A (gpA)+ population, was reduced after co-culture with dexamethasone treated macrophages (25 ±SEM 3.73% CD71+gpA+ erythroblasts) but unaffected by the culture with IL10 polarised cells (42 ±SEM 3.01% CD71+gpA+ erythroblasts). The thesis discovers that macrophages promote erythroblast expansion via inactivation of the transcription factor FOXO3. The presence of either untreated or dexamethasone or IL10 treated macrophages lowered total cellular FOXO3 protein levels in erythroblasts by almost half (0.63 ±SEM 0.02-fold; p=0.0013) while dexamethasone or IL10 modulated macrophages simultaneously increased levels of inactivated phosphorylated-FOXO3 in erythroblasts by 1.39 ±SEM 0.05-fold (p=0.0500) and 1.41 ±SEM 0.04-fold (p=0.0498), respectively. Dexamethasone treated macrophages also elevated levels of the adaptor protein 14-3-3ζ in erythroblasts, suggesting increased export of phosphorylated-FOXO3 to the cytoplasm. However, IL10 treated macrophages did not alter 14-3-3ζ levels, which may indicate that these macrophages did not induce export of phosphorylated FOXO3. The precise role of the macrophage adhesion molecule CD163 was also examined and it was found that siRNA targeted knockdown of this receptor approximately doubled both the total cellular FOXO3 levels and nuclear FOXO3 abundance in erythroblasts (1.81 ±SEM 0.07-fold; p=0.0047 and 2.09 ±SEM 0.21-fold; p=0.0109, respectively, compared to co cultures involving macrophages treated with the control siRNA). The levels of phospho-FOXO3 in contrast were not affected in erythroblasts grown with CD163 deficient macrophages. Analysis of co-cultured supernatants revealed that dexamethasone or IL10 stimulated macrophages had a senescence-associated secretory profile that included CCL2, IL8, Serpin E1, IL1ra and GROα. The addition of IL8 (3,600 pg/ml), Serpin E1 (100 pg/ml) or GROα (110 pg/ml) to erythroblasts cultured alone without macrophages augmented expansion via FOXO3 phosphorylation while GROα simultaneously improved erythroid maturation in a FOXO3-independent manner. The addition to erythroblasts only of all five senescence-associated molecules together at concentrations found in the dexamethasone or IL10 treated macrophage supernatants also elevated erythroid expansion, while the IL10 treated macrophage molecule combinations simultaneously augmented the proportion of mature (CD71+gpA+) erythroid cells by 8 ±SEM 4.18% (p=0.0412). In summary, different in vitro models of macrophage-assisted enhanced erythroblast expansion are provided that either block or preserve erythroid maturation. Furthermore, the contributions of erythroblast macrophage cell-cell contacts and secreted factors are highlighted and an important role for FOXO3 inactivation in erythroid cell expansion is identified. More specifically, the repression of FOXO3 activity is multifactorial, involving both the inhibition of total cellular levels as well as phosphorylation of FOXO3.
Supervisor: Roberts, David J. ; Lamikanra, Abigail Sponsor: National Health Service Blood and Transplant
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Erythropoiesis