Iron is essential for oxygen transport, mitochondrial function, and DNA synthesis, but excess labile iron promotes oxidative stress and tissue dysfunction. As the body lacks a mechanism for iron excretion, iron accumulates with age, yet its impact on skeletal aging remains unclear. We have shown that bone marrow (BM) macrophages progressively accumulate iron in both mice and humans. In aged mice, this is associated with increased CD68⁺ macrophages, but whether this reflects iron-induced dysfunction or a protective sequestration response remains uncertain.

We examined iron metabolism and oxidative stress pathways in young and aged BM macrophages using two complementary approaches. First, targeted immunohistochemistry assessed in situ expression of the iron exporter ferroportin (SLC40A1) and oxidative stress regulators, including GPX4 and SLC7A11/xCT (glutathione pathway), and FSP1 and PRDX1 (glutathione-independent pathways). BM macrophages in both age groups showed robust expression of ferroportin, GPX4, and PRDX1, but lacked macrophage-specific SLC7A11 and FSP1 expression, indicating preserved iron handling and stable oxidative defense mechanisms with aging.

Second, transcriptomic profiling of highly purified BM macrophages (>95% purity, ~40% recovery) supported these findings. Genes for the iron storage complex, ferritin (Ftl1, Fth1), were among the most abundant transcripts, while Slc40a1, Gpx4, and Prdx1 were consistently expressed across ages. In contrast, Slc7a11 and Aifm2 (Fsp1) showed low expression. No broad age-related transcriptional changes in phagocytic or lysosomal pathways were detected. No evidence of glycolytic shift, an expected response to heme (iron) toxicity was observed. However, such changes may not be captured if iron-laden macrophages are under-represented in these datasets.