Background/Objective: Patients with beta-thalassemia are more susceptible to iron overload and have altered neutrophil function. This study investigated the connections between iron metabolism in neutrophils, neutrophil functionality, and overall iron status in individuals with β-thalassemia and sickle cell anemia. Methods: We recruited 18 patients with β-thalassemia, 5 patients with sickle cell anemia, and 15 healthy controls. Our evaluation included measurements of iron and hepcidin concentrations in the serum, along with an analysis of neutrophil function, specifically their phagocytic and oxidative burst capabilities. In addition, we examined the expression of iron transport proteins in neutrophils. Results: Patients with β-thalassemia showed significant iron overload, reduced neutrophil counts, and decreased oxidative burst activity and phagocytosis. Systemic iron status is inversely correlated with the phagocytic capacity of β-thalassemia neutrophils. Regression analysis indicated a significant association between serum iron level, transferrin iron binding capacity, transferrin saturation, and neutrophil percentage. These findings elucidate the essential role of systemic iron levels in neutrophil efficacy against infections. Furthermore, FPN1B and DMT1A mRNA levels were upregulated, and IRP2 was downregulated in the neutrophils of patients with β-thalassemia major and intermedia compared to controls. Conclusions: Elevated systemic iron levels were associated with reduced neutrophil counts and impaired neutrophil function in patients with β-thalassemia. These findings highlight a critical role of systemic iron overload in neutrophil dysfunction.

Aplastic Anemia (AA) arises due to specific failure of bone marrow precursor cells or stem cells to produce adequate amount of mature hematopoietic cells. Erythropoiesis utilizes majority of iron in humans. Erythropoietic activity is fine-tuned with iron regulation since they are both intrinsically regulated. Hepcidin, the main iron regulator is the target of erythropoietic regulators like GDF15, TWSG1 and erythroferrone. Even though the dysregulation of iron balance is well understood in beta thalassemia where ineffective erythropoiesis is the main cause, regulation of iron is poorly elucidated in bone marrow failure where erythropoiesis is minimal. In this study, we analysed several iron regulatory molecules in a cohort of patients with AA and tried to understand iron regulation in bone marrow failure. The current study investigated 77 patients with newly or recently diagnosed AA and had received less than ten transfusions from the year 2016 to 2018. Their haematological parameters were documented. Serum hepcidin was assessed using ELISA kit (DRG Diagnostics) and serum ferritin and soluble transferrin receptor (sTfR) was measured by chemiluminescent immunoassay. DNA was extracted from peripheral blood and telomere length was measured using quantitative real-time PCR (qPCR) using relative quantitation method. The association between different iron regulators were analysed using SPSS Software. The median age of AA patients was 38 (5-72) years and the number of males and females were 49 (64%) and 28 (36%) respectively. Patients were grouped based on severity into non severe AA (NSAA) (n=17, 22%), Severe AA (SAA) (n=51, 66%) and Very Severe AA (VSAA) (n=9, 12%). Out of 77 AA patients, 50 had not received any red cell transfusion in the last one month prior to the sample collection; twenty seven had received transfusions ranging from 1 to 9. The mean haemoglobin was 7.6±1.87g/dL, with a median WBC count of 2900 (100-8400)/cu.mm and a median platelet count of 9000 (2000-66000)/ul. Serum ferritin levels ranged from 12.8 to 5728ng/ml (Median- 1208ng/ml). The median soluble transferrin receptor (sTfR) levels were 1.29mg/L (0.52-6.34). Fifty four patients (54/77, 70%) had sTfR values less than the lower limit of normal (Normal range 1.8-4.6 mg/L). Median serum hepcidin was 40.66ng/ml (1.15-81.2). Serum ferritin levels positively correlated with serum hepcidin (r=0.61, p=0.000) whereas it correlated negatively with sTfR (r=-0.408, p=0.000). No significant association was observed between the number of transfusions and ferritin levels (r=0.087, p=0.453). There was no significant difference in the ferritin levels when patients were grouped according to severity (Median Ferritin, Range - 1208, 12.8-2984ng/ml in NSAA; 1099, 61.8-5728ng/ml in SAA; 1288, 268-3824; p= 0.83). Significantly higher hepcidin levels were observed in VSAA as compared to NSAA (Figure.1, p= 0.014). There was no significant association observed between response and biochemical parameters. A quartile analysis based on ferritin values was carried out to study the effect of iron overload on haematological parameters and iron regulators (Table.1). With increasing ferritin values, hepcidin levels significantly increased (p=0.001) whereas sTfR levels were reduced (p=0.002). In AA, decrease in erythropoietic activity as evidenced by reduced sTfR levels leads to minimal iron utilization and accumulation of iron in the tissues as ferritin. This stimulates the production of hepcidin and further exacerbates iron loading and leads to dysregulation of iron homeostasis. Increased iron in the marrow can lead to oxidative stress and may further be detrimental to the limited intrinsic haematopoiesis. Hence better understanding of iron regulation in AA may advance to effective therapeutic options.

Introduction Beta thalassemia is an inherited disorder characterised by ineffective erythropoiesis leading to anemia and secondary iron overload. Neutrophils are the first line of innate immune defence against infection is dysfunctional in thalassemia patients. Iron is required for the oxidative response of neutrophils to allow the production of reactive oxygen species (ROS). However, the role of iron contributing to the neutrophil dysfunction is unclear. This is the first study to characterise the neutrophil iron metabolism in β-thalassemia and its association with oxidative burst capacity, phagocytosis, and systemic iron homeostasis. Method Sixteen thalassemia patients and fourteen healthy individuals were recruited in the department of Haematology, Christian Medical College, Vellore, India. Neutrophils were purified from human whole blood collected in EDTA tube, using neutrophil magnetic isolation kit (Miltenyi Biotec). Purified population (>95%) was confirmed by the presence of the surface marker CD62L evaluated using flow cytometry. Haematological parameters were analysed according to standard methods. Serum ferritin, iron, soluble transferrin receptor were measured using immunoassay. Serum hepcidin was measured using ELISA. Neutrophil RNA was isolated using trizol method and was reverse transcribed into complementary DNA using QIAGEN kit. The relative quantification of iron related genes were measured using real-time PCR. In oxidative burst assay, neutrophils were incubated with dihydrorhodamine 123 (DHR) and stimulated with Phorbol 12-Myristate 13-Acetate (PMA). Respiratory burst of the cell was analysed by flow cytometry. Phagocytosis and acidification capacity of human neutrophils were quantified using the pHrodo Green Staphylococcus aureus BioParticles kit (Thermo Fisher). Acquisition was performed using the Beckman Coulter(Navios) flow cytometer and analysed using kaluza software. Statistical analysis was performed using SPSS software. Results We investigated a cohort of β thalassemia Major (n=5), intermedia (n=6) and sickle beta thalassemia (n=5) patients who were on regular iron chelation therapy. The demographic and biochemical parameters are tabulated in Table 1. Serum iron, ferritin levels and transferrin saturation were significantly increased in thalassemia cohort as compared to healthy donors (Fig1a). There was no significant association between ferritin and hepcidin levels. The percentage of neutrophils in thalassemia was significantly reduced incomparision to healthy donors (p=0.032). Oxidative burst capacity of neutrophils from thalassemia major and intermedia patients were significantly decreased (p=0.002) compared to healthy donors upon stimulation with PMA (Fig1b). Neutrophil phagocytosis capacity indicated by the relative amounts of phagocytized fluorescein S.aureus particles were significantly lower in thalassemia patients compared with controls, after 15min/30min incubation (Fig1c). The recognition capacity of neutrophils towards bioparticles significantly decreased at 45-minute incubation in patients (p=0.017) (Fig1d). Serum iron overload and transferrin saturation had negative association with neutrophil phagocytosis capacity (r=-0.714; p=0.045 & r=-0.857; p=0.014), respectively. Neutrophil iron related gene expression was analysed and found significantly lower expression of FPN 1A (FPN1 containing iron regulatory element (IRE)), DMT1B without IRE region and IRP2 respectively (p=0.029, p=0.029 & p=0.016) (Fig1e). FPN1B without IRE region was upregulated in thalassemia patients (p=0.016). Serum ferritin had positive correlation with FPN1B (r=0.786; p=0.036). Soluble transferrin receptor had negative association with DMT1A (containing IRE region) and IRP2 respectively (r=-0.900; p=0.037 and r=-0.750; p=0.052). Aberrant neutrophil function was found in all thalassemia patients. Although oxidative burst activity was decreased in thalassemia major and intermedia patients, sickle β-thalassemia patients had normal burst activity. Systemic iron status had inverse correlation with phagocytosis capacity of neutrophils. Dysregulation of iron transporters in neutrophils was indicated by decreased expression of DMT1 and augmented FPN1B expression, despite systemic iron overload. These findings have to be explored in a larger cohort to elucidate the clinical significance.