IMMUNOPHENOTYPTIC ANALYSIS of PBL from beta-THALASSEMIA/HEMOGLOBIN e PATIENTS FOLLOWING HYDROXYUREA THERAPY.

Abstract

Background: Hydroxyurea (HU) is used as a therapeutic option in beta thalassemia to increase fetal hemoglobin, which results in a reduced requirement for blood transfusion. However, a potential adverse effect of HU is neutropenia. Several studies have reported aberrant immune phenotypes and responses in beta thalassemia patients. This raises concerns about the long-term effect of HU therapy with regards to immune function in beta thalassemia patients. Aim(s): This investigate aim to study the immune system of beta-thalassemia/HbE patients following low-dose HU therapy. Method(s): Immuophenotyping analysis was performed on peripheral blood leucocytes (PBL) samples collected from 50 beta-thalassemia/HbE patients. A total of 28 patients received HU treatment (10 mg/kg/day) for more than a year (BE+HU), whereas 22 patients did not receive HU treatment (BE-HU). Peripheral blood was also collected from 26 normal subjects ranging from 18 to 53 years of age. The characterized immunophenotypes, including neutrophils, monocytes, T cells, granulocytic myeloid derived suppressor cells (MDSCs) (G MDSCs) and monocytic-MDSCs (M MDSCs), phagocytosis and oxidative burst activity of peripheral blood neutrophils and monocytes were measured by flow cytometry. In addition, neutrophils were isolated from PBL and the expression of key transcription factors for myeloid maturation and function including C/EBPe, PU.1, C/EBPbeta, C/EBP? and AP1 as markers for neutrophil maturation was determined by RT qPCR. Result(s): The expression of extracellular receptors that are involved in phagocytosis (CD11b, CD16 and CD46; C3bR), inflammation (CD88; C5aR) and migration (CXCR2) and oxidative burst activity were investigated. CD46 expression on neutrophils from BE+HU patients was significantly increased when compared to BE-HU patients, without a significant difference compared with normal subjects. The expression of CD88 and CXCR2 on neutrophils from both groups of patients was significantly decreased as compared with normal subjects. CD4+T cells, CD8+T cells and myeloid derived suppressor cells (G MDSCs and M MDSCs) in BE+HU patients were not significantly different from normal subjects or BE-HU patients. Moreover, Neutrophils and monocytes from both groups of patients had the ability to phagocytose bacteria with no significant difference when compared with normal subjects. Interestingly, the neutrophil and monocyte oxidative burst activity in BE+HU patients increased when compared to BE-HU patients. However, neutrophil transcription factor, such PU.1 and C/EBPbeta, which are associated with neutrophil maturation and function were significantly reduced in BE+HU patients as compared to BE HU patients and normal subjects. Summary/Conclusion: In conclusion, we demonstrate that low-dose HU therapy did not significantly alter immunophenotypic analysis of PBL from BE-HU patients. There was no significant change to neutrophils, monocytes, MDSCs (both granulocytic and monocytic subsets), CD4+T cells, CD8+T cells and complement in this study. Interestingly, we found that low dose HU therapy improved neutrophil oxidative burst activity in BE+HU patients, suggests that low dose HU therapy might be beneficial in diminishing infectious complications. However, we did observe a significantly reduction in the expression of key myeloid transcription factors associated with maturation and function, suggesting HU may influence cellular fates that serve distinct purposes during homeostasis. Further studies are warranted to better understand the long-term effects and efficacy of HU therapy on patients with beta thalassemia and related disorder. (Figure present).