Increased susceptibility of cystic fibrosis airway epithelial cells to ferroptosis

Pramila Maniam,David W Reid,Dora Ling,David M Frazer,Ama-Tawiah Essilfie,Simon Phipps,Gregory J Anderson,Murugan Kalimutho

doi:10.1186/s40659-021-00361-3

Abstract

BackgroundDefective chloride transport in airway epithelial cells (AECs) and the associated lung disease are the main causes of morbidity and early mortality in cystic fibrosis (CF). Abnormal airway iron homeostasis and the presence of lipid peroxidation products, indicative of oxidative stress, are features of CF lung disease.ResultsHere, we report that CF AECs (IB3-1) are susceptible to ferroptosis, a type of cell death associated with iron accumulation and lipid peroxidation. Compared to isogenic CFTR corrected cells (C38), the IB3-1 cells showed increased susceptibility to cell death upon exposure to iron in the form of ferric ammonium citrate (FAC) and the ferroptosis inducer, erastin. This phenotype was accompanied by accumulation of intracellular ferrous iron and lipid peroxides and the extracellular release of malondialdehyde, all indicative of redox stress, and increased levels of lactate dehydrogenase in the culture supernatant, indicating enhanced cell injury. The ferric iron chelator deferoxamine (DFO) and the lipophilic antioxidant ferrostatin-1 inhibited FAC and erastin induced ferroptosis in IB3-1 cells. Glutathione peroxidase 4 (GPX4) expression was decreased in IB3-1 cells treated with FAC and erastin, but was unchanged in C38 AECs. Necroptosis appeared to be involved in the enhanced susceptibility of IB3-1 AECs to ferroptosis, as evidenced by partial cell death rescue with necroptosis inhibitors and enhanced mixed lineage kinase domain-like (MLKL) localisation to the plasma membrane.ConclusionThese studies suggest that the increased susceptibility of CF AECs to ferroptosis is linked to abnormal intracellular ferrous iron accumulation and reduced antioxidant defences. In addition, the process of ferroptotic cell death in CF AECs does not appear to be a single entity and for the first time we describe necroptosis as a potential contributory factor. Iron chelation and antioxidant treatments may be promising therapeutic interventions in cystic fibrosis.Graphical

Highlights

Defective chloride transport in airway epithelial cells (AECs) and the associated lung disease are the main causes of morbidity and early mortality in cystic fibrosis (CF)
IB3‐1 cystic fibrosis cells are more susceptible to cell death by ferroptosis In order to determine the susceptibility of the C38 and IB3-1 cell lines to iron loading, we first measured the levels of intracellular total non-heme iron in the cells
We assessed transferrin receptor 1 (TFR1) expression as an important iron import mechanism to determine whether it may potentially be involved in disrupted cell iron homeostasis as part of ferroptosis, but we found no difference in the expression of TFR1 upon ferric ammonium citrate (FAC) and erastin treatment in either C38 or IB3-1 cells (Additional file 1: Fig. S3)

Summary

Introduction

Defective chloride transport in airway epithelial cells (AECs) and the associated lung disease are the main causes of morbidity and early mortality in cystic fibrosis (CF). Abnormal airway iron homeostasis and the pres‐ ence of lipid peroxidation products, indicative of oxidative stress, are features of CF lung disease. Mutations in CFTR lead to abnormal chloride ion transport in mucus and sweat producing cells, mainly affecting epithelial cells of the respiratory and gastrointestinal tracts [2]. Lung disease with progressive bronchiectasis and airway suppuration remains the main cause of morbidity and premature mortality in patients with CF [3]. Lipid peroxidation products are elevated in CF lungs, suggestive of oxidative stress [5,6,7,8]. Reduced levels of glutathione (GSH), which is a major antioxidant defence in the epithelial lining fluid of the lung have been described, which is probably multifactorial, but primarily related to dysfunctional CFTR that is involved in the extracellular transport of GSH [9,10,11]

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