The Role of Iron-Overloaded Macrophages in Mesenchymal Stem Cell Senescence and Anemia in Myelodysplastic Syndromes: Protocol for an In Vitro Study
Bibliographic record
Abstract
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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.002 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.001 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.007 | 0.003 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".