Haemin-induced cell death in human monocytic cells is consistent with ferroptosis
Introduction
Several diseases render patients to become dependent on regular transfusions, including congenital syndromes, such as thalassemia and sickle cell diseases, and acquired syndromes, such as aplastic anaemia and myelodysplastic syndromes (MDS). Iron overload is a major problem that negatively affects clinical outcome for these transfusion-dependent patients [[1], [2], [3], [4]]. Iron induces cytotoxicity through the production of reactive oxygen species (ROS) [[2], [3], [4]]. Iron accumulation in parenchymal cells, such as hepatocytes, pancreatic beta cells, and cardiac myocytes, causes liver dysfunction, diabetes mellitus, and cardiac failure as the most serious adverse event in such patients [[1], [2], [3], [4]].
The two major sites of iron accumulation are hepatocytes and reticuloendothelial cells (macrophages in the bone marrow and spleen, Kupffer cells in the liver). In hereditary haemochromatosis (HH), a representative disease that causes iron overload, hepatocytes are the first site of iron accumulation [1,2,5]. Deficiency in hepcidin, an iron-regulating hormone, causes uncontrolled iron absorption in enterocytes and iron efflux from macrophages, resulting in elevated plasma iron levels [1,2,5]. Once the transferrin in plasma is saturated with iron, non-transferrin-bound iron (NTBI) is produced, which is taken up by parenchymal cells, and the accumulated iron causes cytotoxicity; however, iron accumulation in macrophages does not occur in HH because of increased iron efflux [1,2,5].
In contrast, reticuloendothelial cells are the first site of iron accumulation in the case of transfusion-related iron overload. Transfusions load large amounts of iron on macrophages as haem-iron [1,3,4]. Macrophages phagocytose senescent and damaged red blood cells (RBCs) and take up haemoglobin released from RBCs and haem released from extracellular haemoglobin [6]. Haem is catabolized by haem oxygenase-1 (HO-1) into iron, carbon monoxide, and biliverdin, and the processed iron is stored in cytoplasmic ferritin [1,2]. Selective accumulation of iron in reticuloendothelial cells is relatively safe and protects parenchymal cells from iron overload [1]. When the amount of loaded iron exceeds the macrophage’s protective capacity, iron accumulates in parenchymal cells and causes organ dysfunction similar to HH [1,2]. Therefore, macrophages play a major role in the pathophysiology of transfusion iron overload.
Despite recognition of the clinical relevance of haem-iron overload in macrophages during transfusion iron overload, there have been few detailed investigations of these specific effects and mechanisms conducted to date [[7], [8], [9], [10], [11], [12]].
Bozza’s research group has intensively studied the underlying factors driving haem-related cytotoxicity using murine peritoneal macrophages and revealed that haem induces non-apoptotic cell death and ROS production via two pathways: iron-mediated ROS production and toll-like receptor 4 (TLR4)-mediated tumour necrosis factor (TNF) production [[8], [9], [10]]. Moreover, Bozza’s group further demonstrated the iron-dependence of this haem-induced macrophage cell death [8,9], which led us hypothesize that this effect is an example of ferroptosis, an iron-dependent non-apoptotic cell death mechanism originally proposed by Stockwell et al. in 2012 [13]. A hallmark of ferroptosis is iron-dependent lipid peroxidation, which can be inhibited by iron chelators, and peroxidation inhibitors (i.e. ferroptosis inhibitors) such as ferrostatins and liproxstatins [[13], [14], [15], [16]]. The haem-induced macrophage cell death described by Bozza’s group for murine peritoneal macrophages appears to be largely consistent with the known process of ferroptosis, except for the dependence on TLR4 pathway. Indeed, we previously demonstrated that haemin induces non-apoptotic cell death and ROS generation in human monocytic THP-1 cells [17]. Haemin is the oxidized form of haem and the major component of extracellular haem in vivo [6].
In the present study, to test our hypothesis that haemin-induced cell death is an example of ferroptosis, we examined the effects of a ferroptosis inhibitor and inducer on haemin-induced THP-1 cell death.
Section snippets
Cell culture
Human monocytic THP-1 cells, purchased from DS Pharma Biomedical (Osaka, Japan), were maintained in RPMI1640 medium supplemented with 10% foetal bovine serum (FBS; Hyclone, South Logan, Utah, USA) in a 5% CO2 atmosphere at 37 °C.
Treatment with haemin and other reagents
THP-1 cells were washed with phosphate-buffered saline (PBS) twice to remove the FBS. The cells were then suspended in RPMI1640 medium at a density of 1 × 106 cells/ml and 1 ml of the cell suspension was seeded in each well of a 24-well plate. Haemin (Sigma, St. Louis,
Haemin induced cell death and ROS generation in a dose-dependent manner
To examine the cytotoxic effects of haemin, THP-1 cells were treated with 0–20 μmol/l of haemin for 2 h under a serum-free condition. After haemin treatment, the proportion of cells exhibiting a necrotic pattern (Annexin-V– PI+) increased, but there was no change in the proportion of cells showing an apoptotic pattern (Annexin-V + PI+/–) (Fig. 1a, b). In addition, the percentages of viable (Annexin-V– PI–) cells decreased after haemin treatment in a dose-dependent manner (Fig. 1c).
The level of
Discussion
In transfusion iron overload, loading of haem-iron occurs through two major routes: endocytosis of extracellular haem and phagocytosis of RBCs.
Extracellular haem acts as a damage-associated molecular pattern and causes inflammatory responses in monocyte/macrophages and neutrophils [3,[6], [7], [8], [9], [10],[18], [19], [20], [21], [22]]. Macrophages produce inflammatory cytokines, chemokines, and lipid mediators [6,[7], [8], [9], [10],18]. According to the work of Bozza’s group, haem induces
Authors’ contributions
S.I., M.K. and K.S. designed the research experiments; S.I., Y.S., T.Sa., Y.M. and A.O. performed the experiments; S.I., T.Su. and H.S. analyzed the data; S.I. wrote the paper.
Funding
This work was supported by Kobe Tokiwa University Research Fund (2015–2017).
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgements
We thank Yuiko Ando, Yuka Tamura, Rika Tanaka, Anri Fukudome, and Risaho Fujioka for technical assistance with laboratory work.
We would also like to thank Editage (www.editage.jp) for English language editing.
References (24)
- et al.
Impact of iron overload and potential benefit from iron chelation in low-risk myelodysplastic syndrome
Blood
(2014) - et al.
Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins
Blood
(2013) - et al.
Macrophages and iron metabolism
Immunity
(2016) - et al.
Heme induces programmed necrosis on macrophages through autocrine TNF and ROS production
Blood
(2012) - et al.
Characterization of heme as activator of Toll-like receptor 4
J Biol Chem
(2007) - et al.
Macrophages and iron trafficking at the birth and death of red cells
Blood
(2015) - et al.
Ferroptosis: an iron-dependent form of nonapoptotic cell death
Cell
(2012) - et al.
Heme-induced neutrophil extracellular traps contribute to the pathogenesis of sickle cell disease
Blood
(2014) - et al.
Quantitative analysis of hemin-induced neutrophil extracellular trap formation and effects of hydrogen peroxide on this phenomenon
Biochem Biophys Rep
(2017) - et al.
Intracellular labile iron pools as direct targets of iron chelators: a fluorescence study of chelator action in living cells
Blood
(2005)
Clinical relevance of anemia and transfusion iron overload in myelodysplastic syndromes
Hematology Am Soc Hematol Educ Program
Body iron metabolism and pathophysiology of iron overload
Int J Hematol
Cited by (46)
Impact of ferroptosis on preeclampsia: A review
2023, Biomedicine and PharmacotherapyFerroptosis in life: To be or not to be
2023, Biomedicine and PharmacotherapyFerroptosis-related small-molecule compounds in cancer therapy: Strategies and applications
2022, European Journal of Medicinal ChemistryCross-link between type 2 diabetes mellitus and iron deficiency anemia. A mini-review
2022, Clinical Nutrition Open ScienceCitation Excerpt :This results in impaired Hb synthesis, ineffective hemopoiesis, and thus iron deficiency anemia. Consistently, in case the iron levels are excessively elevated, they accumulate in the bone marrow and hematopoietic cells compartment resulting in the generation of reactive oxygen species (ROS), thus damaging hematopoietic cells [62,63]. Moreover, increased iron levels in the blood due to multiple blood transfusions result in iron overload, damaging organs, and thus leading to liver cirrhosis [62,64].
Labile iron, ROS, and cell death are prominently induced by haemin, but not by non-transferrin-bound iron
2022, Transfusion and Apheresis ScienceCitation Excerpt :Iron-dependent cell death is thought to be ferroptosis [1,7–9]. We previously reported that haemin-induced cell death of monocytes/macrophages is consistent with ferroptosis [10,11]. However, haem/haemin is thought to be a damage-associated molecular pattern (DAMP) and may induce pro-inflammatory cytokines [12–14].