Non-Transferrin-Bound Iron Drives Ferroptosis in THP-1 derived Macrophages via Heme oxygenase-1 Pathway

Transfusion-induced iron overload presents a major clinical challenge, often leading to progressive organ damage and increased vulnerability to infections, suggesting an underlying immune cell dysfunction. Macrophages are central to iron metabolism and are highly susceptible to the cytotoxic effects of non-transferrin-bound iron (NTBI) accumulation. This study investigated the mechanisms of NTBI-induced cytotoxicity in THP-1 derived macrophages (TDMs) with a specific focus on the regulatory role of the heme oxygenase-1 (HO-1) signaling pathway. We demonstrated that NTBI induces dose- and time-dependent cell death in TDMs primarily through ferroptosis, characterized by increased lipid peroxidation and dependence on extracellular iron uptake. While the HO-1 pathway is activated by iron, we uncovered its paradoxical role: HO-1 acts as a perpetrator of ferroptosis. Inhibition of HO-1 significantly rescued cell viability and attenuated lipid peroxidation, an effect directly linked to the blockade of the release of HO-1-catalyzed ferrous iron (Fe ²⁺ ) into the labile iron pool. Crucially, this protective effect occurred without restoring the protein levels of glutathione peroxidase 4 (GPX4), suggesting a GPX4-independent mechanism for HO-1-mediated toxicity. Our findings redefine the function of the Nrf2-HO-1 axis in iron-overloaded macrophages. By proving that HO-1’s pro-oxidant function dominates under iron saturation, our work highlights HO-1 as a complex but novel therapeutic target for mitigating iron-induced pathologies and the associated immune dysfunction.