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

Non-Transferrin-Bound Iron Drives Ferroptosis in THP-1 derived Macrophages via Heme oxygenase-1 Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Non-Transferrin-Bound Iron Drives Ferroptosis in THP-1 derived Macrophages via Heme oxygenase-1 Pathway Shih-Chung Wang, Cheng-Han Lee, Kan-Hsuan Lin, Rei-Cheng Yang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8072729/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. Ferroptosis Heme oxygenase-1 (HO-1) THP-1 macrophages (TDMs) Iron overload Non-Transferrin-Bound Iron (NTBI) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Transfusion-induced iron overload is a significant clinical concern, particularly in patients with transfusion-dependent thalassemia. Regular blood transfusions lead to iron accumulation, causing progressive organ damage and potentially fatal complications(Betts et al., 2020 ; Taher & Saliba, 2017 ). Importantly, these patients also exhibit increased vulnerability to bacterial infections, suggesting a potential link between disrupted iron regulation and immune dysfunction(Farmakis et al., 2022 ; Ganz, 2018 ). Within the plasma, iron is primarily transported and bound to transferrin. However, when iron levels exceed the transferrin binding capacity, non-transferrin-bound iron (NTBI) appears, leading to its accumulation in various cell types(Knutson, 2019 ). This intracellular iron excess triggers cytotoxicity through the production of reactive oxygen species (ROS), which often culminates in regulated cell death pathways such as apoptosis and, more recently recognized and intensely studied, ferroptosis. This process ultimately leads to organ damage and cell death (Galaris et al., 2019 ). Iron homeostasis is a complex process that is regulated by various molecules to maintain optimal iron levels, including absorption, utilization, recycling by the reticuloendothelial system, and storage(Silva & Faustino, 2015 ). Macrophages are key components of the immune system and are particularly susceptible to iron overload owing to their central role in iron metabolism and storage(Korolnek & Hamza, 2015 ; Recalcati & Cairo, 2021 ). These cells are crucial for maintaining a delicate balance between iron availability for biological functions and prevention of cytotoxic effects(Soares & Hamza, 2016 ). The intricate interplay between macrophages and iron has significant implications for immune function and various diseases including infections and atherosclerosis(Nairz et al., 2015 ). Recent studies have highlighted the relationship between intracellular iron accumulation and ferroptosis, a newly characterized form of regulated cell death that is driven by iron-dependent lipid peroxidation and ROS generation(Dixon et al., 2012 ; Yang et al., 2022 ). Ferroptosis is distinct from other forms of cell death and is characterized by the accumulation of lipid hydroperoxides, leading to membrane damage(Li et al., 2020 ). This unique modality is intricately controlled by various cellular metabolic pathways, including those governing redox balance, iron processing, mitochondrial function, and metabolism of amino acids, lipids, and sugars, along with relevant signaling pathways(Jiang et al., 2021 ). As macrophages are essential for host defense, ferroptosis due to iron overload may contribute to the observed increase in susceptibility to infections (Jiang et al., 2024 ). Imoto et al. demonstrated that hemin, a heme-derived source of iron, induces significant ROS production and cell death in THP-1 cells through mechanisms consistent with ferroptosis(Imoto et al., 2018 ). However, whether NTBI directly drives ferroptosis in macrophages remains unclear. The Nrf2-HO-1 pathway is integral to the management of oxidative stress and susceptibility to ferroptosis (Dodson et al., 2019 ; Yan et al., 2023 ). Heme oxygenase-1 (HO-1), the enzyme at the end of this cascade, breaks down heme, producing biliverdin, carbon monoxide, and ferrous iron (Fe 2+) . While generally considered a protective antioxidant molecule that supports cell viability (Zhang et al., 2020 ), HO-1 has recently been implicated in the initiation of cell death. Because the degradation of heme directly supplies Fe 2+ , a high level of HO-1 expression in an already iron-saturated environment can facilitate ferroptosis, effectively switching its role from protective to pro-death (Chang et al., 2018 ; Chiang et al., 2018 ). Consequently, cellular concentrations of iron and ROS determine the functional outcome of HO-1. In this study, we demonstrated that NTBI induces dose- and time-dependent cell death in THP-1 derived macrophages (TDMs), which is primarily executed via ferroptosis, driven by extracellular iron uptake. Crucially, we uncovered the paradoxical role of the Nrf2-HO-1 axis, showing that iron-induced HO-1 acts as a mediator of cell death. HO-1 inhibition rescues viability and lipid peroxidation, a protective effect mediated by blocking the HO-1 catalyzed release of Fe 2+ into the labile iron pool, a mechanism independent of glutathione peroxidase 4 (GPX4) regulation. Our findings redefine the role of HO-1 in iron-overloaded macrophages and highlight a novel therapeutic target for preventing iron-induced immune dysfunction. Materials and Methods Reagents and antibodies: Ferrous sulfate heptahydrate (FeSO₄·7H₂O), Phorbol 12-myristate 13-acetate (PMA), 3-Methyladenine (3-MA), and Z-VAD-FMK were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tin Protoporphyrin IX (SnPPIX) and Ferrostatin-1 were obtained from Cayman Chemical (Ann Arbor, MI, USA). FerroOrange for ferrous iron detection was acquired from Dojindo Laboratories (F374, Kumamoto, Japan). BODIPY™ 581/591 C11, was from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). The following primary antibodies were used for western blot analysis: LC3 (NB100-2220, 1:1000) from Novus Biologicals (Littleton, CO, USA); Cleaved-caspase-3 (9661, 1:1000), HO-1 (43966, 1:1000), and Nrf2 (12721, 1:1000) from Cell Signaling Technology (Danvers, MA, USA); SLC7A11/xCT (ARG57998, 1:1000) and GPX4 (ARG41400, 1:1000) from Arigo Biolaboratories Co. (Hsinchu City, Taiwan); and β-actin (sc-47778, 1:10000) from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell culture and treatments: Human monocytic leukemia THP-1 cells (ATCC TIB-202) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 IU/mL penicillin G, 100 µg/mL streptomycin, and 2 mM L-glutamine. The cells were maintained at 37°C in a humidified 5% CO₂ atmosphere. For differentiation into macrophages, THP-1 monocytes were seeded at a density of 5×10 5 cells/well in 24-well plates and treated with 25 ng/mL PMA for 48 h. After differentiation, the medium was removed, and cells were washed once with PBS and rested in fresh PMA-free medium for 24 h before any experiments. Differentiated TDMs were treated with varying concentrations of FeSO₄ for up to 48 hours. For inhibition experiments, cells were pre-treated for 1 h with the following compounds before co-treatment with 10 mM FeSO₄: pan-caspase inhibitor Z-VAD-FMK (20 µM), autophagy inhibitor 3-MA (1 mM), Nrf2 inhibitor ML385 (10 µM), endocytosis inhibitor Cytochalasin D (5 µM), ferroptosis inhibitor, Ferrostatin-1 (60 µM), and HO-1 inhibitor SnPPIX (0–10 µM). Nrf2 Gene Silencing: Nrf2 gene silencing was performed by transfecting TDMs with 50 nM Nrf2-specific siRNA (s9491, Thermo Fisher Scientific, Waltham, MA, USA) or non-targeting control siRNA (4390843, Thermo Fisher Scientific) using Lipofectamine™ 3000 (Thermo Fisher Scientific). After a 36-hour transfection period, the medium was refreshed, and cells were subsequently treated with or without 10 mM FeSO₄ for 24 hours. Cell Viability Assessment: MTT assay (M5655, Sigma, St. Louis, MO, USA) was used to assess cell viability. Following treatments, 0.5 mg/mL MTT solution was added to each well and incubated for 4 h at 37°C. The formazan crystals were dissolved in DMSO and the absorbance was measured at 570 nm using a microplate reader (Thermo Multiskan SPECTRUM, Thermo Fisher Scientific, Waltham, MA, USA). Apoptosis Analysis: Apoptosis was measured via flow cytometry using Annexin V and Propidium Iodide (PI) staining (Cat. No.640914, Biolegend, UK). After treatment, the cells were collected, suspended in Annexin V Binding Buffer, and incubated for 20 min with Annexin V and PI. Data were acquired using a Cytomics FC500 Flow Cytometer (Beckman-Coulter, Villepinte, France). Reactive Oxygen Species (ROS) Detection: The intracellular ROS levels were evaluated by flow cytometry using a DHR123 fluorescent probe (Cat. No. D1054, Sigma, USA). Cells were incubated with 5 µM DHR123 for 30 min, washed, and analyzed using a Cytomics™ FC500 Flow Cytometer. Lipid Peroxidation Measurement: Lipid peroxidation was assessed using a BODIPY 581/591 C11 probe. Cells were incubated with 10 µM of the probe for 20 min, and green fluorescence resulting from oxidation was detected using a fluorescence microscope (FV1000, Olympus, Hamburg, Germany). Intracellular Ferrous Iron (Fe²⁺) Assessment: The intracellular Fe²⁺ levels were assessed using a FerroOrange fluorescent probe. Following treatment, cells were incubated with 5 µM FerroOrange for 30 min. Cellular fluorescence was detected and quantified using fluorescence microscopy and flow cytometry. Western blot analysis: Cells were lysed in RIPA buffer (97063-270, VWR, Radnor, PA, USA) supplemented with a Protease Inhibitor Cocktail (11836170001; Roche, Basel, Switzerland). Protein concentrations were determined using Bradford assay. Samples (10 µg of protein) were separated by SDS-PAGE on 10% or 12% polyacrylamide gels. The proteins were transferred to PVDF membranes by electroblotting. Membranes were blocked with 5% non-fat dry milk in 1x TBS-T and incubated overnight at 4°C with primary antibodies. HRP-conjugated secondary antibodies(GTX221667-01 or GTX213110-01; Genetex Hsinchu, Taiwan ) were used at a 1:5000 dilution for 1 h. Protein bands were visualized using a chemiluminescence imaging analyzer, and densitometric quantification was performed using the ImageJ software and normalized to β-actin. Statistical Analysis All experiments were conducted with at least three independent biological replicates, and each assay was performed in duplicate or triplicate. Data are presented as mean ± Standard Error of the Mean (SEM). Statistical analyses were performed using SPSS Statistics, and differences between two groups were analyzed using Student's t-test. A p -value of less significance was set at P < 0.05. Results NTBI Induces Dose- and Time-Dependent Cytotoxicity and Apoptosis in TDMs To determine the cytotoxic effects of NTBI on macrophages, TDMs were exposed to FeSO 4 (0–10 mM) for 24 and 48 h. The MTT assay revealed that iron treatment caused dose- and time-dependent reductions in TDM viability (Fig. 1 A). A significant decrease in viability was observed starting at 10 mM FeSO 4 at both time points. Consequently, 10 mM FeSO 4 was selected as the standard concentration for all the subsequent mechanistic experiments. To assess the role of apoptosis, TDMs treated with 10 mM FeSO 4 were analyzed by flow cytometry using Annexin V/Propidium Iodide (PI) staining. After 48 h of iron treatment, approximately 35% of the cell population showed Annexin V/PI positivity, confirming the induction of apoptotic cell death (Fig. 1 B). Western blot analysis revealed a time-dependent increase in the protein levels of the apoptotic marker cleaved-caspase-3 after exposure to 10 mM FeSO 4 from 16 to 48 h (Fig. 1 C). To confirm the functional contribution of this pathway, TDMs were pre-treated with the pan-caspase inhibitor Z-VAD-FMK (20 µM). This pre-treatment provided partial but significant protection against FeSO 4 -induced cell death, validating that apoptosis contributes to overall toxicity (Fig. 1 D). Autophagy does not modulate iron-induced cytotoxicity in TDMs To explore the potential involvement of autophagy in iron-induced cytotoxicity, expression of key autophagic markers was examined. Western blot analysis showed that FeSO 4 treatment led to time-dependent conversion of LC3-I to its lipidated form, LC3-II, along with a gradual decrease in the protein level of p62 (Fig. 2 A). These findings collectively suggest robust initiation and flux of the autophagic pathway following iron treatment. To determine if this activation functionally modulated cell death, TDMs were pretreated with the specific autophagy inhibitor, 3-Methyladenine (3-MA, 1 mM). Unlike the partial protection observed with the caspase inhibitor, 3-MA failed to rescue TDMs and showed no significant change in cell viability compared with the iron-only group (Fig. 2 B). These results suggest that, while autophagy is activated by iron, it does not appear to directly modulate cell viability or contribute to the final cytotoxic outcome in TDMs. Ferroptosis is a Key Mechanism of NTBI-induced Cell Death, Characterized by Increased Intracellular Iron and ROS Following assessment of apoptosis and autophagy, the potential involvement of ferroptosis in iron-induced TDM cell death was investigated. Consistent with the hallmarks of ferroptosis, iron treatment led to a substantial increase in lipid peroxidation, quantified using the C11-BODIPY probe (Fig. 3 A). To confirm the functional significance of this finding, pre-treatment with Ferrostatin-1 (60 µM), a specific ferroptosis inhibitor, significantly rescued TDMs from cell death induced by 10 mM FeSO 4 (Fig. 3 B), providing direct evidence for ferroptosis involvement. Further characterization revealed that iron treatment led to a substantial increase in intracellular ferrous iron (Fe 2+ ) levels, as demonstrated by the FerroOrange analysis (Fig. 3 C), as well as significantly elevated ROS generation, as measured by the DHR 123 assay (Fig. 3 D). To confirm the mechanism of iron accumulation, the effects of Cytochalasin D, an inhibitor of actin-dependent endocytosis, were examined. Cytochalasin D treatment significantly reduced the intracellular ferrous iron content following FeSO 4 exposure (Fig. 3 E), indicating that the increased iron was predominantly taken up from the extracellular environment via active cellular processes. To definitively exclude ferritin degradation (ferritinophagy) as a contributor to the observed increase in iron, the key components of this pathway were assessed. Western blot analysis revealed an increase in ferritin protein levels and a decrease in NCOA4 expression (Fig. 3 F). This expression pattern is contrary to that expected during active ferritinophagy, which strongly suggests that the elevated intracellular iron originates predominantly from extracellular non-heme iron uptake rather than from the degradation of intracellular ferritin stores. Finally, to characterize the execution phase of ferroptosis, key regulators were assessed by western blotting. The primary anti-ferroptotic enzyme, GPX4, was markedly decreased, whereas the expression of the cystine/glutamate antiporter, SLC7A11(xCT), remained unchanged (Fig. 3 G). These findings collectively confirm that NTBI-induced cell death in TDMs is mediated by ferroptosis driven by extracellular iron uptake. Iron activates Nrf2-HO-1 Signaling Axis in TDMs Given the established role of the Nrf2-HO-1 axis in cellular responses to oxidative stress and iron, its involvement in iron-induced TDM cytotoxicity was investigated. Western blot analysis revealed a time-dependent increase in the ratio of phosphorylated Nrf2 (pNrf2) to total Nrf2 following FeSO 4 treatment, indicating Nrf2 activation (Fig. 4 A). Concurrently, HO-1 protein levels were markedly upregulated during the early stage of FeSO₄ treatment (peaking around 24 h) and gradually declined thereafter (Fig. 4 A). To confirm the functional relationship between Nrf2 and HO-1 in this context, siRNA-mediated knockdown of Nrf2 (siNrf2) was employed. This intervention resulted in a significant reduction in HO-1 expression following iron treatment (Fig. 4 B). Collectively, these findings demonstrate that FeSO 4 treatment effectively activates the Nrf2-HO-1 signaling pathway in TDMs, with Nrf2 phosphorylation acting upstream to regulate HO-1 expression. HO-1 Inhibition Attenuates Iron-Induced Lipid Peroxidation and Rescues TDMs Viability To investigate the functional significance of HO-1 upregulation in ferroptosis, the HO-1 inhibitor, SnPPIX, was used. Treatment with SnPPIX significantly attenuated the increase in lipid peroxidation observed in iron-treated TDMs, as quantified using the C11-BODIPY probe (Fig. 5 A). Furthermore, SnPPIX remarkably rescued TDM cell viability, which was compromised by iron exposure, demonstrating a clear dose-dependent protective effect (Fig. 5 B). These findings strongly indicate that HO-1 promotes iron-induced cell death. To probe the mechanism of this pro-ferroptotic role, the effect of SnPPIX on key markers was assessed. Although SnPPIX provided clear protection, it did not significantly alter the protein levels of GPX4 in iron-treated TDMs (Fig. 5 C). This suggests that HO-1 contributes to ferroptosis in TDMs through a mechanism independent of GPX4 regulation. Further analysis showed that SnPPIX treatment decreased intracellular ferrous iron levels (Fig. 5 D) but caused no significant change in the overall intracellular ROS level (Fig. 5 E). Discussion This study aimed to elucidate the cellular mechanisms by which iron overload induces cytotoxicity in TDMs. Our findings revealed a multifaceted cell death response: while apoptosis is a contributing factor, ferroptosis is the prominent mode of cell death in response to iron overload (Jiang et al., 2024). Although autophagy was activated, its inhibition failed to rescue cell viability, suggesting a nonessential role in determining cell fate in this context. This research significantly advances our understanding of iron toxicity by identifying the key drivers of ferroptosis in TDMs. We showed that the process is initiated by the robust uptake of extracellular NTBI (FeSO 4 ), which fuels the labile iron pool and drives ROS production and subsequent lipid peroxidation. Crucially, this dependence on extracellular NTBI uptake rather than the degradation of intracellular ferritin stores (ferritinophagy) represents a critical mechanistic insight specific to macrophages, which are uniquely specialized in iron handling (Duca et al., 2025; Fibach & Rachmilewitz, 2017). This mechanism also bypasses canonical ferroptosis initiation pathways, such as System Xc - inhibition (Chen et al., 2020). The most striking finding is the pivotal yet paradoxical role of the Nrf2-HO-1 axis (Chiang et al., 2021; de Oliveira et al., 2022; Yan et al., 2023). Despite its typical cytoprotective function, we demonstrated that iron-induced activation of this pathway mediates ferroptosis. This transition is directly to HO-1’s enzymatic activity: in the setting of sustained iron overload, the HO-1-catalyzed breakdown of heme releases highly reactive ferrous iron (Fe 2+ ), which directly contributes to the labile iron pool and amplifies the lethal Fenton reaction(Chang et al., 2018; Chiang et al., 2018; Garcia-Santos et al., 2018; Hassannia et al., 2018). This pro-ferroptotic mechanism is strongly supported by functional data from HO-1 inhibition with SnPPIX. SnPPIX treatment significantly decreased intracellular Fe 2+ levels, confirming that HO-1 is a source of cytotoxic iron. Furthermore, while SnPPIX rescued viability and inhibited lipid peroxidation, it did not restore GPX4 protein levels or significantly alter bulk intracellular ROS levels. This suggests that HO-1 does not increase generalized oxidative stress or regulate GPX4, but rather specifically fuels the localized lipid peroxidation cascade via Fe 2+ delivery in a GPX4-independent manner. This model is fundamentally tied to macrophage biology, where their specialization in iron handling makes them uniquely susceptible to the consequences of HO-1 activation under iron saturation. This also provides a plausible explanation for the discrepancy with previous studies showing limited NTBI effects in monocytic THP-1 cell lines, suggesting distinct iron-handling capabilities between monocytes and fully differentiated TDMs (Haschka et al., 2019; Imoto et al., 2022; Mesquita et al., 2020) . Although our study provides novel mechanistic insights, it has limitations, primarily stemming from the use of an in vitro cell line model, which lacks the complexity of an in vivo environment (Tedesco et al., 2018). Furthermore, the acute, high-dose FeSO 4 model used here may not perfectly reflect the slow, chronic iron accumulation observed in patients (Fu & Yang, 2025; Yadav & Singh, 2022). Finally, the precise mechanistic crosstalk between apoptosis and ferroptosis requires further elucidation (Eskander et al., 2025; Wu et al., 2023). Our findings open several promising avenues for future research, primarily to bridge the gap in clinical relevance. Validating the paradoxical HO-1 in an animal model of iron overload (e.g., a thalassemia mouse model) (Lob et al., 2025; Sanyear et al., 2020) is a crucial next step, which would allow for testing the therapeutic potential of targeting HO-1 to mitigate iron-induced organ damage (Garcia-Santos et al., 2018; Nithichanon et al., 2020). This finding carries significant translational implications, suggesting that in iron-overload conditions, pharmacological inhibition of HO-1 could be a viable and targeted therapeutic strategy to mitigate tissue damage associated with conditions like transfusion-dependent thalassemia. Further exploration of the downstream signaling cascades that link HO-1’s catalytic activity to the amplification of lipid peroxidation remains essential. Collectively, these future studies will build upon our foundational work and guide the development of novel therapeutic strategies for iron-induced pathologies. Declarations Funding Declaration This research was supported by grants from Changhua Christian Hospital (grant numbers 110-CCH-IRP-096 and 110-CCH-IRP-081), the National Chung Hsing University/Changhua Christian Hospital Joint Research Program (NCHU-CCH-11201), and the National Science and Technology Council, Taiwan (MOST 111-2320-B-005-003-MY3). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Clinical trial number: not applicable Author Contribution SCW, CHL, KHL, JJS and JKK developed the initial ideas; formulated the research goals and aims. SCW, CHL, KHL, RCY, CSH, JJS, JKK designed the research methods and experimental procedures. CHL, JJS and JKK verified the results or experiments; ensured the accuracy of the data. SCW, RCY, CHL, CSH applied statistical, mathematical, or computational techniques to analyze data. SCW, CHL, RCY, KHL, CSH, JJS, JKK conducted the research and carried out the experiments. SCW, JJS, CHL managed, cleaned, and maintained research data. SCW wrote the first version of the manuscript. JJS and JKK critically reviewed and revised the manuscript. SCW, RCY, CHL and CSH created figures, charts, or other visual representations of the data. JJS and JKK managed and coordinated the research activity. All authors read and approved the final manuscript. References Betts, M., Flight, P. A., Paramore, L. C., Tian, L., Milenkovic, D., & Sheth, S. (2020). Systematic Literature Review of the Burden of Disease and Treatment for Transfusion-dependent beta-Thalassemia. 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16:05:44","extension":"png","order_by":77,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":63000,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8072729/v1/a8ac561b14e5f1d6f0dddaae.png"},{"id":99307749,"identity":"14d8fd8c-8818-4c55-b114-c235a06d6157","added_by":"auto","created_at":"2025-12-31 16:06:42","extension":"png","order_by":78,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":97008,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8072729/v1/f2f2f623cfaf0c950bfeceb1.png"},{"id":98812960,"identity":"a06a0718-79cd-4245-960c-05fb22fa0b80","added_by":"auto","created_at":"2025-12-22 15:49:14","extension":"png","order_by":79,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":607,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8072729/v1/4f9d89948461cf19b7d8f207.png"},{"id":99307344,"identity":"28c42a45-10c7-4bd2-891c-a37891acdf95","added_by":"auto","created_at":"2025-12-31 16:06:03","extension":"xml","order_by":80,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":138448,"visible":true,"origin":"","legend":"","description":"","filename":"1a96eba6afd84032877a840939aa5e851structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8072729/v1/eeffa4fbb9f828b88f560040.xml"},{"id":99307812,"identity":"723f8fb9-fdff-40a0-b73f-de4040d8fb9e","added_by":"auto","created_at":"2025-12-31 16:06:51","extension":"html","order_by":81,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":157548,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8072729/v1/45ecbfa6c8e8bed5bbb50c84.html"},{"id":98812882,"identity":"8c2c4d41-567e-4595-8973-68b649e45bad","added_by":"auto","created_at":"2025-12-22 15:49:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":228796,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIron induces cytotoxicity and apoptosis in TDMs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Cell viability of TDMs was assessed by MTT assay after exposure to varying concentrations of iron (FeSO\u003csub\u003e4\u003c/sub\u003e) for 24 and 48 hours. \u003cstrong\u003e(B)\u003c/strong\u003e Representative flow cytometry plots and quantification of apoptotic TDMs following iron treatment, assessed by Annexin V/Propidium Iodide (PI) staining. \u003cstrong\u003e(C)\u003c/strong\u003e Time-dependent protein expression of the apoptotic marker, cleaved-caspase-3, in TDMs following iron treatment was analyzed by Western blot. \u003cstrong\u003e(D)\u003c/strong\u003e Cell viability was assessed by MTT assay after TDMs were treated with iron in the presence or absence of the pan-caspase inhibitor, Z-VAD-FMK, to confirm the contribution of apoptosis to cell death. Data are presented as means ± SEM from three independent experiments. Statistical significance is indicated as follows: *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8072729/v1/44e237a63ab7d9bf0529ff4a.png"},{"id":98812885,"identity":"f9def7b9-41f6-4f0d-b00e-9dbe7fd36bb4","added_by":"auto","created_at":"2025-12-22 15:49:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":207566,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIron Activates Autophagy in TDMs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Time-dependent protein expression of autophagic markers, specifically the conversion of LC3-I to LC3-II, in TDMs following iron treatment, analyzed by Western blot. \u003cstrong\u003e(B)\u003c/strong\u003e Cell viability was assessed by MTT assay after TDMs were co-treated with iron in the presence or absence of the autophagy inhibitor, 3-Methyladenine (3-MA). Data are presented as means ± SEM from 3 independent experiments. Statistical significance is indicated as follows: *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8072729/v1/b377dbe530055f18c1ee39c9.png"},{"id":98812883,"identity":"72a31a52-0e1c-4114-a6a7-82190ecbdca9","added_by":"auto","created_at":"2025-12-22 15:49:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":304155,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIron Induces Ferroptosis in TDMs via Extracellular Iron Uptake\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Quantification of lipid peroxidation in TDMs following iron treatment, assessed using the C11-BODIPY probe. \u003cstrong\u003e(B)\u003c/strong\u003e The functional role of ferroptosis was confirmed by assessing cell viability (MTT assay) after TDMs were co-treated with iron in the presence or absence of the specific ferroptosis inhibitor, Ferrostatin-1. \u003cstrong\u003e(C)\u003c/strong\u003e Quantification of intracellular ferrous iron in TDMs following iron treatment, assessed using the fluorescent probe FerroOrange and analyzed by fluorescence microscopy. \u003cstrong\u003e(D)\u003c/strong\u003e Quantification of intracellular ROS in TDMs following iron treatment, assessed using the fluorescent probe DHR 123 and analyzed by flow cytometry. \u003cstrong\u003e(E)\u003c/strong\u003eIntracellular ferrous iron levels were assessed by the FerroOrange probe in TDMs treated with iron in the presence or absence of Cytochalasin D, an endocytosis inhibitor, and analyzed by fluorescence microscopy. \u003cstrong\u003e(F)\u003c/strong\u003e Representative Western blot analysis and corresponding densitometric quantification showing the protein levels of NCOA4 and ferritin in TDMs after iron treatment. Protein quantification was normalized to β-actin. \u003cstrong\u003e(G)\u003c/strong\u003e Representative Western blot analysis and corresponding densitometric quantification showing the protein levels of GPX4, and SLC7A11 in TDMs following iron treatment. Protein quantification was normalized to β-actin. Data are presented as means ± SEM from three independent experiments. Statistical significance is indicated as follows: *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001; ****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8072729/v1/07f1a14c6ac467291c9aa41e.png"},{"id":98812887,"identity":"cd02bbdc-73b4-4857-901c-f309fcbf0250","added_by":"auto","created_at":"2025-12-22 15:49:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":185231,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIron Activates the Nrf2-HO-1 Axis in TDMs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Representative Western blot images and corresponding densitometric quantification showing the protein levels of total Nrf2, phosphorylated Nrf2 (pNrf2), and HO-1 in TDMs following a time course of iron treatment (16 to 48 hours). Quantification panels show the ratio of pNrf2 to total Nrf2 and the relative level of HO-1. All protein levels are normalized to β-actin. \u003cstrong\u003e(B)\u003c/strong\u003e Western blot analysis and quantification of HO-1 protein expression after siRNA-mediated knockdown of Nrf2 (siNrf2) in the presence or absence of iron treatment, demonstrating that iron-induced HO-1 expression is dependent on Nrf2.Data are presented as means ± SEM from three independent experiments. Statistical significance is indicated as follows: *\u003cem\u003ep \u003c/em\u003e\u0026lt;0.05; **\u003cem\u003ep \u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003ep \u003c/em\u003e\u0026lt;0.001; ****\u003cem\u003ep \u003c/em\u003e\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8072729/v1/89f73bc0f93bb70fbbd1e2cf.png"},{"id":99307063,"identity":"b498dea3-5b0a-4f8b-90d9-a461d2621d87","added_by":"auto","created_at":"2025-12-31 16:05:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":232242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHO-1 Inhibition Rescues TDMs Cell Viability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Quantification of lipid peroxidation in TDMs following iron treatment (10 mM FeSO\u003csub\u003e4\u003c/sub\u003e) with or without the inhibitor, SnPPIX at varying concentrations, assessed using the C11-BODIPY probe. \u003cstrong\u003e\u0026nbsp;(B)\u003c/strong\u003e Cell viability assessed by MTT assay after TDMs were treated with iron in the presence or absence of SnPPIX at varying concentrations (0, 1, 2, 6, 8, 10 μM) to demonstrate the dose-dependent effect. \u003cstrong\u003e(C)\u003c/strong\u003e Representative Western blot analysis and densitometric quantification of protein level of the key anti-ferroptotic enzyme, GPX4, in TDMs treated with iron and/or SnPPIX. \u003cstrong\u003e(D)\u003c/strong\u003e Intracellular ferrous iron (Fe\u003csup\u003e2+\u003c/sup\u003e) levels assessed by the FerroOrange probe in TDMs treated with iron and/or SnPPIX.\u003cstrong\u003e (E)\u003c/strong\u003e Quantification of overall intracellular ROS in TDMs treated with iron and/or SnPPIX, assessed using the DHR 123 probe. Data are presented as means ± SEM from three independent experiments. Statistical significance is indicated as follows: *\u003cem\u003ep \u003c/em\u003e\u0026lt;0.05; **\u003cem\u003ep \u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003ep \u003c/em\u003e\u0026lt;0.001; ****\u003cem\u003ep \u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8072729/v1/b52ea8180532c8a298114b24.png"},{"id":99307083,"identity":"dcb75ebd-704a-4581-a672-426bb0f5d396","added_by":"auto","created_at":"2025-12-31 16:05:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":522010,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of the mechanism of ferroptosis induced by iron overload\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8072729/v1/6e7a4f262788a120a9ec6762.png"},{"id":99801245,"identity":"0f3cb0e5-93c8-4247-8f50-14b065563c9c","added_by":"auto","created_at":"2026-01-08 14:04:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2412021,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8072729/v1/7bf16130-b138-4eec-b0dd-008671d28057.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Non-Transferrin-Bound Iron Drives Ferroptosis in THP-1 derived Macrophages via Heme oxygenase-1 Pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTransfusion-induced iron overload is a significant clinical concern, particularly in patients with transfusion-dependent thalassemia. Regular blood transfusions lead to iron accumulation, causing progressive organ damage and potentially fatal complications(Betts et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Taher \u0026amp; Saliba, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Importantly, these patients also exhibit increased vulnerability to bacterial infections, suggesting a potential link between disrupted iron regulation and immune dysfunction(Farmakis et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ganz, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Within the plasma, iron is primarily transported and bound to transferrin. However, when iron levels exceed the transferrin binding capacity, non-transferrin-bound iron (NTBI) appears, leading to its accumulation in various cell types(Knutson, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This intracellular iron excess triggers cytotoxicity through the production of reactive oxygen species (ROS), which often culminates in regulated cell death pathways such as apoptosis and, more recently recognized and intensely studied, ferroptosis. This process ultimately leads to organ damage and cell death (Galaris et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIron homeostasis is a complex process that is regulated by various molecules to maintain optimal iron levels, including absorption, utilization, recycling by the reticuloendothelial system, and storage(Silva \u0026amp; Faustino, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Macrophages are key components of the immune system and are particularly susceptible to iron overload owing to their central role in iron metabolism and storage(Korolnek \u0026amp; Hamza, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Recalcati \u0026amp; Cairo, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These cells are crucial for maintaining a delicate balance between iron availability for biological functions and prevention of cytotoxic effects(Soares \u0026amp; Hamza, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The intricate interplay between macrophages and iron has significant implications for immune function and various diseases including infections and atherosclerosis(Nairz et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecent studies have highlighted the relationship between intracellular iron accumulation and ferroptosis, a newly characterized form of regulated cell death that is driven by iron-dependent lipid peroxidation and ROS generation(Dixon et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Ferroptosis is distinct from other forms of cell death and is characterized by the accumulation of lipid hydroperoxides, leading to membrane damage(Li et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This unique modality is intricately controlled by various cellular metabolic pathways, including those governing redox balance, iron processing, mitochondrial function, and metabolism of amino acids, lipids, and sugars, along with relevant signaling pathways(Jiang et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As macrophages are essential for host defense, ferroptosis due to iron overload may contribute to the observed increase in susceptibility to infections (Jiang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Imoto et al. demonstrated that hemin, a heme-derived source of iron, induces significant ROS production and cell death in THP-1 cells through mechanisms consistent with ferroptosis(Imoto et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, whether NTBI directly drives ferroptosis in macrophages remains unclear.\u003c/p\u003e \u003cp\u003eThe Nrf2-HO-1 pathway is integral to the management of oxidative stress and susceptibility to ferroptosis (Dodson et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yan et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Heme oxygenase-1 (HO-1), the enzyme at the end of this cascade, breaks down heme, producing biliverdin, carbon monoxide, and ferrous iron (Fe\u003csup\u003e2+)\u003c/sup\u003e. While generally considered a protective antioxidant molecule that supports cell viability (Zhang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), HO-1 has recently been implicated in the initiation of cell death. Because the degradation of heme directly supplies Fe\u003csup\u003e2+\u003c/sup\u003e, a high level of HO-1 expression in an already iron-saturated environment can facilitate ferroptosis, effectively switching its role from protective to pro-death (Chang et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Chiang et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Consequently, cellular concentrations of iron and ROS determine the functional outcome of HO-1.\u003c/p\u003e \u003cp\u003eIn this study, we demonstrated that NTBI induces dose- and time-dependent cell death in THP-1 derived macrophages (TDMs), which is primarily executed via ferroptosis, driven by extracellular iron uptake. Crucially, we uncovered the paradoxical role of the Nrf2-HO-1 axis, showing that iron-induced HO-1 acts as a mediator of cell death. HO-1 inhibition rescues viability and lipid peroxidation, a protective effect mediated by blocking the HO-1 catalyzed release of Fe\u003csup\u003e2+\u003c/sup\u003e into the labile iron pool, a mechanism independent of glutathione peroxidase 4 (GPX4) regulation. Our findings redefine the role of HO-1 in iron-overloaded macrophages and highlight a novel therapeutic target for preventing iron-induced immune dysfunction.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents and antibodies:\u003c/h2\u003e \u003cp\u003eFerrous sulfate heptahydrate (FeSO₄\u0026middot;7H₂O), Phorbol 12-myristate 13-acetate (PMA), 3-Methyladenine (3-MA), and Z-VAD-FMK were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tin Protoporphyrin IX (SnPPIX) and Ferrostatin-1 were obtained from Cayman Chemical (Ann Arbor, MI, USA). FerroOrange for ferrous iron detection was acquired from Dojindo Laboratories (F374, Kumamoto, Japan). BODIPY\u0026trade; 581/591 C11, was from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). The following primary antibodies were used for western blot analysis: LC3 (NB100-2220, 1:1000) from Novus Biologicals (Littleton, CO, USA); Cleaved-caspase-3 (9661, 1:1000), HO-1 (43966, 1:1000), and Nrf2 (12721, 1:1000) from Cell Signaling Technology (Danvers, MA, USA); SLC7A11/xCT (ARG57998, 1:1000) and GPX4 (ARG41400, 1:1000) from Arigo Biolaboratories Co. (Hsinchu City, Taiwan); and β-actin (sc-47778, 1:10000) from Santa Cruz Biotechnology (Santa Cruz, CA, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture and treatments:\u003c/h3\u003e\n\u003cp\u003eHuman monocytic leukemia THP-1 cells (ATCC TIB-202) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 IU/mL penicillin G, 100 \u0026micro;g/mL streptomycin, and 2 mM L-glutamine. The cells were maintained at 37\u0026deg;C in a humidified 5% CO₂ atmosphere. For differentiation into macrophages, THP-1 monocytes were seeded at a density of 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well in 24-well plates and treated with 25 ng/mL PMA for 48 h. After differentiation, the medium was removed, and cells were washed once with PBS and rested in fresh PMA-free medium for 24 h before any experiments.\u003c/p\u003e \u003cp\u003eDifferentiated TDMs were treated with varying concentrations of FeSO₄ for up to 48 hours. For inhibition experiments, cells were pre-treated for 1 h with the following compounds before co-treatment with 10 mM FeSO₄: pan-caspase inhibitor Z-VAD-FMK (20 \u0026micro;M), autophagy inhibitor 3-MA (1 mM), Nrf2 inhibitor ML385 (10 \u0026micro;M), endocytosis inhibitor Cytochalasin D (5 \u0026micro;M), ferroptosis inhibitor, Ferrostatin-1 (60 \u0026micro;M), and HO-1 inhibitor SnPPIX (0\u0026ndash;10 \u0026micro;M).\u003c/p\u003e\n\u003ch3\u003eNrf2 Gene Silencing:\u003c/h3\u003e\n\u003cp\u003eNrf2 gene silencing was performed by transfecting TDMs with 50 nM Nrf2-specific siRNA (s9491, Thermo Fisher Scientific, Waltham, MA, USA) or non-targeting control siRNA (4390843, Thermo Fisher Scientific) using Lipofectamine\u0026trade; 3000 (Thermo Fisher Scientific). After a 36-hour transfection period, the medium was refreshed, and cells were subsequently treated with or without 10 mM FeSO₄ for 24 hours.\u003c/p\u003e\n\u003ch3\u003eCell Viability Assessment:\u003c/h3\u003e\n\u003cp\u003eMTT assay (M5655, Sigma, St. Louis, MO, USA) was used to assess cell viability. Following treatments, 0.5 mg/mL MTT solution was added to each well and incubated for 4 h at 37\u0026deg;C. The formazan crystals were dissolved in DMSO and the absorbance was measured at 570 nm using a microplate reader (Thermo Multiskan SPECTRUM, Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e\n\u003ch3\u003eApoptosis Analysis:\u003c/h3\u003e\n\u003cp\u003eApoptosis was measured via flow cytometry using Annexin V and Propidium Iodide (PI) staining (Cat. No.640914, Biolegend, UK). After treatment, the cells were collected, suspended in Annexin V Binding Buffer, and incubated for 20 min with Annexin V and PI. Data were acquired using a Cytomics FC500 Flow Cytometer (Beckman-Coulter, Villepinte, France).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eReactive Oxygen Species (ROS) Detection:\u003c/h2\u003e \u003cp\u003eThe intracellular ROS levels were evaluated by flow cytometry using a DHR123 fluorescent probe (Cat. No. D1054, Sigma, USA). Cells were incubated with 5 \u0026micro;M DHR123 for 30 min, washed, and analyzed using a Cytomics\u0026trade; FC500 Flow Cytometer.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLipid Peroxidation Measurement:\u003c/h3\u003e\n\u003cp\u003eLipid peroxidation was assessed using a BODIPY 581/591 C11 probe. Cells were incubated with 10 \u0026micro;M of the probe for 20 min, and green fluorescence resulting from oxidation was detected using a fluorescence microscope (FV1000, Olympus, Hamburg, Germany).\u003c/p\u003e\n\u003ch3\u003eIntracellular Ferrous Iron (Fe²⁺) Assessment:\u003c/h3\u003e\n\u003cp\u003eThe intracellular Fe\u0026sup2;⁺ levels were assessed using a FerroOrange fluorescent probe. Following treatment, cells were incubated with 5 \u0026micro;M FerroOrange for 30 min. Cellular fluorescence was detected and quantified using fluorescence microscopy and flow cytometry.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis:\u003c/h2\u003e \u003cp\u003eCells were lysed in RIPA buffer (97063-270, VWR, Radnor, PA, USA) supplemented with a Protease Inhibitor Cocktail (11836170001; Roche, Basel, Switzerland). Protein concentrations were determined using Bradford assay. Samples (10 \u0026micro;g of protein) were separated by SDS-PAGE on 10% or 12% polyacrylamide gels. The proteins were transferred to PVDF membranes by electroblotting. Membranes were blocked with 5% non-fat dry milk in 1x TBS-T and incubated overnight at 4\u0026deg;C with primary antibodies. HRP-conjugated secondary antibodies(GTX221667-01 or GTX213110-01; Genetex Hsinchu, Taiwan ) were used at a 1:5000 dilution for 1 h. Protein bands were visualized using a chemiluminescence imaging analyzer, and densitometric quantification was performed using the ImageJ software and normalized to β-actin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll experiments were conducted with at least three independent biological replicates, and each assay was performed in duplicate or triplicate. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard Error of the Mean (SEM). Statistical analyses were performed using SPSS Statistics, and differences between two groups were analyzed using Student's t-test. A \u003cem\u003ep\u003c/em\u003e-value of less significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eNTBI Induces Dose- and Time-Dependent Cytotoxicity and Apoptosis in TDMs\u003c/h2\u003e \u003cp\u003eTo determine the cytotoxic effects of NTBI on macrophages, TDMs were exposed to FeSO\u003csub\u003e4\u003c/sub\u003e (0\u0026ndash;10 mM) for 24 and 48 h. The MTT assay revealed that iron treatment caused dose- and time-dependent reductions in TDM viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). A significant decrease in viability was observed starting at 10 mM FeSO\u003csub\u003e4\u003c/sub\u003e at both time points. Consequently, 10 mM FeSO\u003csub\u003e4\u003c/sub\u003e was selected as the standard concentration for all the subsequent mechanistic experiments.\u003c/p\u003e \u003cp\u003eTo assess the role of apoptosis, TDMs treated with 10 mM FeSO\u003csub\u003e4\u003c/sub\u003e were analyzed by flow cytometry using Annexin V/Propidium Iodide (PI) staining. After 48 h of iron treatment, approximately 35% of the cell population showed Annexin V/PI positivity, confirming the induction of apoptotic cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Western blot analysis revealed a time-dependent increase in the protein levels of the apoptotic marker cleaved-caspase-3 after exposure to 10 mM FeSO\u003csub\u003e4\u003c/sub\u003e from 16 to 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). To confirm the functional contribution of this pathway, TDMs were pre-treated with the pan-caspase inhibitor Z-VAD-FMK (20 \u0026micro;M). This pre-treatment provided partial but significant protection against FeSO\u003csub\u003e4\u003c/sub\u003e-induced cell death, validating that apoptosis contributes to overall toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAutophagy does not modulate iron-induced cytotoxicity in TDMs\u003c/h2\u003e \u003cp\u003eTo explore the potential involvement of autophagy in iron-induced cytotoxicity, expression of key autophagic markers was examined. Western blot analysis showed that FeSO\u003csub\u003e4\u003c/sub\u003e treatment led to time-dependent conversion of LC3-I to its lipidated form, LC3-II, along with a gradual decrease in the protein level of p62 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These findings collectively suggest robust initiation and flux of the autophagic pathway following iron treatment. To determine if this activation functionally modulated cell death, TDMs were pretreated with the specific autophagy inhibitor, 3-Methyladenine (3-MA, 1 mM). Unlike the partial protection observed with the caspase inhibitor, 3-MA failed to rescue TDMs and showed no significant change in cell viability compared with the iron-only group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These results suggest that, while autophagy is activated by iron, it does not appear to directly modulate cell viability or contribute to the final cytotoxic outcome in TDMs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFerroptosis is a Key Mechanism of NTBI-induced Cell Death, Characterized by Increased Intracellular Iron and ROS\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFollowing assessment of apoptosis and autophagy, the potential involvement of ferroptosis in iron-induced TDM cell death was investigated. Consistent with the hallmarks of ferroptosis, iron treatment led to a substantial increase in lipid peroxidation, quantified using the C11-BODIPY probe (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To confirm the functional significance of this finding, pre-treatment with Ferrostatin-1 (60 \u0026micro;M), a specific ferroptosis inhibitor, significantly rescued TDMs from cell death induced by 10 mM FeSO\u003csub\u003e4\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), providing direct evidence for ferroptosis involvement.\u003c/p\u003e \u003cp\u003eFurther characterization revealed that iron treatment led to a substantial increase in intracellular ferrous iron (Fe\u003csup\u003e2+\u003c/sup\u003e) levels, as demonstrated by the FerroOrange analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), as well as significantly elevated ROS generation, as measured by the DHR 123 assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTo confirm the mechanism of iron accumulation, the effects of Cytochalasin D, an inhibitor of actin-dependent endocytosis, were examined. Cytochalasin D treatment significantly reduced the intracellular ferrous iron content following FeSO\u003csub\u003e4\u003c/sub\u003e exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), indicating that the increased iron was predominantly taken up from the extracellular environment via active cellular processes.\u003c/p\u003e \u003cp\u003eTo definitively exclude ferritin degradation (ferritinophagy) as a contributor to the observed increase in iron, the key components of this pathway were assessed. Western blot analysis revealed an increase in ferritin protein levels and a decrease in NCOA4 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). This expression pattern is contrary to that expected during active ferritinophagy, which strongly suggests that the elevated intracellular iron originates predominantly from extracellular non-heme iron uptake rather than from the degradation of intracellular ferritin stores.\u003c/p\u003e \u003cp\u003eFinally, to characterize the execution phase of ferroptosis, key regulators were assessed by western blotting. The primary anti-ferroptotic enzyme, GPX4, was markedly decreased, whereas the expression of the cystine/glutamate antiporter, SLC7A11(xCT), remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). These findings collectively confirm that NTBI-induced cell death in TDMs is mediated by ferroptosis driven by extracellular iron uptake.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eIron activates Nrf2-HO-1 Signaling Axis in TDMs\u003c/h2\u003e \u003cp\u003eGiven the established role of the Nrf2-HO-1 axis in cellular responses to oxidative stress and iron, its involvement in iron-induced TDM cytotoxicity was investigated. Western blot analysis revealed a time-dependent increase in the ratio of phosphorylated Nrf2 (pNrf2) to total Nrf2 following FeSO\u003csub\u003e4\u003c/sub\u003e treatment, indicating Nrf2 activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Concurrently, HO-1 protein levels were markedly upregulated during the early stage of FeSO₄ treatment (peaking around 24 h) and gradually declined thereafter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eTo confirm the functional relationship between Nrf2 and HO-1 in this context, siRNA-mediated knockdown of Nrf2 (siNrf2) was employed. This intervention resulted in a significant reduction in HO-1 expression following iron treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Collectively, these findings demonstrate that FeSO\u003csub\u003e4\u003c/sub\u003e treatment effectively activates the Nrf2-HO-1 signaling pathway in TDMs, with Nrf2 phosphorylation acting upstream to regulate HO-1 expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHO-1 Inhibition Attenuates Iron-Induced Lipid Peroxidation and Rescues TDMs Viability\u003c/h2\u003e \u003cp\u003eTo investigate the functional significance of HO-1 upregulation in ferroptosis, the HO-1 inhibitor, SnPPIX, was used. Treatment with SnPPIX significantly attenuated the increase in lipid peroxidation observed in iron-treated TDMs, as quantified using the C11-BODIPY probe (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Furthermore, SnPPIX remarkably rescued TDM cell viability, which was compromised by iron exposure, demonstrating a clear dose-dependent protective effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These findings strongly indicate that HO-1 promotes iron-induced cell death.\u003c/p\u003e \u003cp\u003eTo probe the mechanism of this pro-ferroptotic role, the effect of SnPPIX on key markers was assessed. Although SnPPIX provided clear protection, it did not significantly alter the protein levels of GPX4 in iron-treated TDMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). This suggests that HO-1 contributes to ferroptosis in TDMs through a mechanism independent of GPX4 regulation. Further analysis showed that SnPPIX treatment decreased intracellular ferrous iron levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) but caused no significant change in the overall intracellular ROS level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study aimed to elucidate the cellular mechanisms by which iron overload induces cytotoxicity in TDMs. Our findings revealed a multifaceted cell death response: while apoptosis is a contributing factor, ferroptosis is the prominent mode of cell death in response to iron overload (Jiang et al., 2024). Although autophagy was activated, its inhibition failed to rescue cell viability, suggesting a nonessential role in determining cell fate in this context. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis research significantly advances our understanding of iron toxicity by identifying the key drivers of ferroptosis in TDMs. We showed that the process is initiated by the robust uptake of extracellular NTBI (FeSO\u003csub\u003e4\u003c/sub\u003e), which fuels the labile iron pool and drives ROS production and subsequent lipid peroxidation. Crucially, this dependence on extracellular NTBI uptake rather than the degradation of intracellular ferritin stores (ferritinophagy) represents a critical mechanistic insight specific to macrophages, which are uniquely specialized in iron handling (Duca et al., 2025; Fibach \u0026amp; Rachmilewitz, 2017). This mechanism also bypasses canonical ferroptosis initiation pathways, such as System\u0026nbsp;Xc\u003csup\u003e-\u003c/sup\u003e inhibition (Chen et al., 2020).\u003c/p\u003e\n\u003cp\u003eThe most striking finding is the pivotal yet paradoxical role of the Nrf2-HO-1 axis (Chiang et al., 2021; de Oliveira et al., 2022; Yan et al., 2023). Despite its typical cytoprotective function, we demonstrated that iron-induced activation of this pathway mediates ferroptosis. This transition is directly to HO-1’s enzymatic activity: in the setting of sustained iron overload, the HO-1-catalyzed breakdown of heme releases highly reactive ferrous iron (Fe\u003csup\u003e2+\u003c/sup\u003e), which directly contributes to the labile iron pool and amplifies the lethal Fenton reaction(Chang et al., 2018; Chiang et al., 2018; Garcia-Santos et al., 2018; Hassannia et al., 2018). This pro-ferroptotic mechanism is strongly supported by functional data from HO-1 inhibition with SnPPIX. SnPPIX treatment significantly decreased intracellular Fe\u003csup\u003e2+\u003c/sup\u003e levels, confirming that HO-1 is a source of cytotoxic iron. Furthermore, while SnPPIX rescued viability and inhibited lipid peroxidation, it did not restore GPX4 protein levels or significantly alter bulk intracellular ROS levels. This suggests that HO-1 does not increase generalized oxidative stress or regulate GPX4, but rather specifically fuels the localized lipid peroxidation cascade via Fe\u003csup\u003e2+\u003c/sup\u003e delivery in a GPX4-independent manner.\u003c/p\u003e\n\u003cp\u003eThis model is fundamentally tied to macrophage biology, where their specialization in iron handling makes them uniquely susceptible to the consequences of HO-1 activation under iron saturation. This also provides a plausible explanation for the discrepancy with previous studies showing limited NTBI effects in monocytic THP-1 cell lines, suggesting distinct iron-handling capabilities between monocytes and fully differentiated TDMs (Haschka et al., 2019; Imoto et al., 2022; Mesquita et al., 2020) .\u003c/p\u003e\n\u003cp\u003eAlthough our study provides novel mechanistic insights, it has limitations, primarily stemming from the use of an in vitro cell line model, which lacks the complexity of an \u003cem\u003ein vivo\u003c/em\u003e environment (Tedesco et al., 2018). Furthermore, the acute, high-dose FeSO\u003csub\u003e4\u003c/sub\u003e model used here may not perfectly reflect the slow, chronic iron accumulation observed in patients (Fu \u0026amp; Yang, 2025; Yadav \u0026amp; Singh, 2022). Finally, the precise mechanistic crosstalk between apoptosis and ferroptosis requires further elucidation (Eskander et al., 2025; Wu et al., 2023).\u003c/p\u003e\n\u003cp\u003eOur findings open several promising avenues for future research, primarily to bridge the gap in clinical relevance. Validating the paradoxical HO-1 in an animal model of iron overload (e.g., a thalassemia mouse model) (Lob et al., 2025; Sanyear et al., 2020) is a crucial next step, which would allow for testing the therapeutic potential of targeting HO-1 to mitigate iron-induced organ damage (Garcia-Santos et al., 2018; Nithichanon et al., 2020). This finding carries significant translational implications, suggesting that in iron-overload conditions, pharmacological inhibition of HO-1 could be a viable and targeted therapeutic strategy to mitigate tissue damage associated with conditions like transfusion-dependent thalassemia. Further exploration of the downstream signaling cascades that link HO-1’s catalytic activity to the amplification of lipid peroxidation remains essential. Collectively, these future studies will build upon our foundational work and guide the development of novel therapeutic strategies for iron-induced pathologies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by grants from Changhua Christian Hospital (grant numbers 110-CCH-IRP-096 and 110-CCH-IRP-081), the National Chung Hsing University/Changhua Christian Hospital Joint Research Program (NCHU-CCH-11201), and the National Science and Technology Council, Taiwan (MOST 111-2320-B-005-003-MY3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003e not applicable\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSCW, CHL, KHL, JJS and JKK developed the initial ideas; formulated the research goals and aims. SCW, CHL, KHL, RCY, CSH, JJS, JKK designed the research methods and experimental procedures. CHL, JJS and JKK verified the results or experiments; ensured the accuracy of the data. SCW, RCY, CHL, CSH applied statistical, mathematical, or computational techniques to analyze data. SCW, CHL, RCY, KHL, CSH, JJS, JKK conducted the research and carried out the experiments. SCW, JJS, CHL managed, cleaned, and maintained research data. SCW wrote the first version of the manuscript. JJS and JKK critically reviewed and revised the manuscript. SCW, RCY, CHL and CSH created figures, charts, or other visual representations of the data. JJS and JKK managed and coordinated the research activity. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBetts, M., Flight, P. A., Paramore, L. C., Tian, L., Milenkovic, D., \u0026amp; Sheth, S. (2020). 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The Nrf-2/HO-1 Signaling Axis: A Ray of Hope in Cardiovascular Diseases. \u003cem\u003eCardiology Research and Practice\u003c/em\u003e, \u003cem\u003e2020\u003c/em\u003e, 5695723. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2020/5695723\u003c/span\u003e\u003cspan address=\"10.1155/2020/5695723\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ferroptosis, Heme oxygenase-1 (HO-1), THP-1 macrophages (TDMs), Iron overload, Non-Transferrin-Bound Iron (NTBI)","lastPublishedDoi":"10.21203/rs.3.rs-8072729/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8072729/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTransfusion-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\u003csup\u003e2+\u003c/sup\u003e) 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\u0026rsquo;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.\u003c/p\u003e","manuscriptTitle":"Non-Transferrin-Bound Iron Drives Ferroptosis in THP-1 derived Macrophages via Heme oxygenase-1 Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-22 15:49:06","doi":"10.21203/rs.3.rs-8072729/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9958a082-38b8-4e89-967e-a19948a980d5","owner":[],"postedDate":"December 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-04T22:08:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-22 15:49:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8072729","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8072729","identity":"rs-8072729","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}