The Role of Iron-Overloaded Macrophages in Mesenchymal Stem Cell Senescence and Anemia in Myelodysplastic Syndromes: Protocol for an In Vitro Study

BACKGROUND: Myelodysplastic syndromes (MDSs) are a group of highly heterogeneous myeloid clonal diseases. Anemia is the most common clinical symptom, yet its pathogenesis remains incompletely understood. Preliminary evidence suggests an increase in macrophage infiltration and iron load in the bone marrow of patients with an MDS, alongside elevated interleukin-6 (IL-6) expression in bone marrow mesenchymal stem cells (BMSCs). The Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is a critical regulator of cellular antioxidant responses and inflammation, but its role in mediating the effects of iron overload in the microenvironment of patients with an MDS remains unclear. OBJECTIVE: This study aims to investigate the hypothesis that iron-overloaded bone marrow macrophages promote BMSC senescence and IL-6 secretion via the Keap1-Nrf2-ARE pathway, thereby impairing the survival and differentiation of hematopoietic stem cells and hematopoietic progenitor cells and contributing to anemia. Specific objectives are to (1) quantify the macrophage iron load across major MDS subtypes and correlate it with anemia severity and prognosis; (2) define the phenotype of erythroid island macrophages (CD68+, CD169+, and vascular cell adhesion molecule 1+) and the expression of endothelial microparticles in bone marrow tissues of patients with an MDS; and (3) experimentally test whether iron-overloaded macrophages and their exosomes regulate IL-6 secretion and senescence in BMSCs via the Keap1-Nrf2-ARE pathway. METHODS: Bone marrow samples will be collected from patients with key MDS subtypes (MDS with single-lineage dysplasia, refractory anemia with ring sideroblasts, MDS with multilineage dysplasia, and MDS with excess blasts; n=30 per subgroup) and control patients with iron-deficiency anemia (n=30). Methods will include histochemistry (Perls Prussian blue staining), immunohistochemistry and immunofluorescence (for macrophage and endothelial microparticle analysis), enzyme-linked immunosorbent assay, flow cytometry, quantitative polymerase chain reaction, and Western blotting. An in vitro model of iron-overloaded macrophages will be established using phorbol 12-myristate 13-acetate-differentiated THP-1 cells treated with ferric ammonium citrate. Exosomes will be isolated from these macrophages via ultracentrifugation. The effects of iron-overloaded macrophages and their exosomes on BMSC IL-6 secretion, senescence (senescence-associated β-galactosidase staining), and Keap1-Nrf2-ARE pathway activity will be assessed in coculture systems, with and without pharmacological inhibitors (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole) or activators (dimethyl fumarate) of Nrf2. RESULTS: The study received ethics approval in December 2024. Patient recruitment and sample collection are in progress. As of December 2025, a total of 85 samples had been accrued. Pilot experiments to optimize macrophage differentiation and iron loading conditions have been completed. The full experimental workflow, including all sample analyses and in vitro experiments, is anticipated to be completed by May 2026. CONCLUSIONS: This study is expected to elucidate a novel molecular mechanism linking iron overload in macrophages to BMSC dysfunction and anemia in MDS. The findings could identify the Keap1-Nrf2-ARE pathway as a potential therapeutic target for managing MDS-related anemia. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/77936.