Investigating the role of GDF15 in β-thalassemia

Abstract

β-thalassemia is a group of heterogeneous hereditary disorders characterised by reduced or absent β-globin chain synthesis. A primary cause of β-thalassemia disease pathophysiology is ineffective erythropoiesis (IE), involving increased expansion of erythroid progenitors, and increased apoptosis during erythroid differentiation. GDF15 levels are high in the serum of patients with β-thalassemia and have been directly correlated with disease severity and indirectly with the degree of ineffective
erythropoiesis. However, there is little direct evidence for this or the role of GDF15 in causing defective erythroid cell differentiation. Due to the limited availability of suitable patient material for in vitro studies, this project initially focused on establishing a cellular system and characterising the most suitable β-thalassemia erythroid cell line for investigating the role of GDF15 in IE.
The adult human erythroid cell line, BEL-A and a β-thalassemia model line created by CRISPR-Cas9 knockout of the HBB genes (HBB-/-) in BEL-A, were used to investigate the detrimental effect of GDF15 on IE. Primary cells from normal individuals and a β-thalassemia patient were included to validate our results. Our data indicate that GDF15 levels increased during differentiation, correlating with progression to the polychromatic stage, and were higher in β-thalassemia cells compared to
control erythroid cells. Additionally, neither IE induction in control, nor exacerbation of IE in β-thalassemia cells was found when these cells were treated with recGDF15. Therefore, we considered an alternative role proposed in a previous study for GDF15 in the pathophysiology of β-thalassemia, which is inhibition of hepcidin expression. For this we added recGDF15 to cultures of the human hepatocyte line HuH-7. However, there was no effect of GDF15 on hepcidin expression. This led us to
explore the receptor for GDF15, glial-derived neurotrophic factor (GDNF)-family receptor α-like (GFRAL) in erythroid cells and hepatocytes. The receptor was not detected, supporting our data showing no effect of GDF15 on these cells. Finally, we examined the possibility that the effect of GDF15 in previous studies was due to contamination of the recGDF15 by TGF-β. Indeed, addition of TGF-β1 to HuH-7 cells suppressed hepcidin expression. Our results suggest the proposed roles for GDF15 in β-thalassemia may instead have been caused by TGF-β1.
To definitively determine whether GDF15 plays an intrinsic role of GDF15 in erythroid
differentiation, the GDF15 gene was knocked out in both WT BEL-A and HBB-/- cells. Our findings refute the notion of a detrimental effect of GDF15 in β-thalassemia; however, its absence induced apoptosis in erythroid cells. Overall, the work described in this thesis confirms that GDF15 is not a causative factor in the differentiation defects of β-thalassemia erythroid cells but may serve as an
anti-apoptotic factor.

Date of Award	4 Feb 2025
Original language	English
Awarding Institution	University of Bristol
Supervisor	Jan Frayne (Supervisor) & Belinda K Singleton (Supervisor)