Iron Buildup May Lead to Death of CF Airway Cells, Study Suggests
Airway cells in cystic fibrosis (CF) were more susceptible to death due to the buildup of iron and their reduced antioxidant ability, a study suggested.
These findings support further research into the use of iron-binding chelators and antioxidants as potential CF therapies, the scientists said.
The study, “Increased susceptibility of cystic fibrosis airway epithelial cells to ferroptosis,” was published in the journal Biological Research.
In CF, a faulty CFTR protein leads to abnormal transport of chloride ions in mucus- and sweat-producing epithelial cells that line the airways and digestive tract. As a result, people with CF produce thick, sticky mucus and experience ongoing infections, inflammation, and tissue damage.
Excess iron, or iron that is not stored safely within cells, can trigger the production of harmful reactive oxygen species (ROS), known as free radicals. This, in turn, promotes the toxic buildup of lipid peroxides — breakdown products of fat and fatlike substances within cells — promoting an iron-dependent mechanism of cell death called ferroptosis.
Although there is growing evidence for the impact of ferroptosis in lung diseases such as asthma and chronic obstructive pulmonary disease (COPD), ferroptosis in CF remains poorly understood.
In this report, researchers at the QIMR Berghofer Medical Research Institute in Australia “investigated the involvement and mechanism of ferroptosis in CF airway epithelial cells (AECs),” the team wrote.
Epithelial cells were derived from a CF patient, and healthy cells were created by introducing a functioning copy of the CFTR protein.
Both cell lines were found to have the same total iron levels, even after iron treatment with ferric ammonium citrate (FAC), a compound that releases iron. However, in the presence of erastin, a small molecule that blocks production of the antioxidant glutathione (GSH), CF cells had significantly more iron levels than healthy cells.
In both cells lines, erastin alone did not decrease cell viability. Still, treatment with iron and erastin led to a dose-dependent decrease in CF cell viability, as compared with non-CF cells. Higher levels of lactate dehydrogenase (LDH) — a marker for cellular damage — were also seen in treated CF cells.
“Taken together, these data demonstrate that [CF cells] have increased susceptibility to cell death by ferroptosis,” the researchers wrote.
To confirm these results, cells were exposed to iron and erastin in combination with the iron chelator DFO, which binds iron and prevents ROS generation, and the antioxidant Fer-1, an inhibitor of toxic lipid peroxide formation. Consistently, DFO and Fer-1 prevented cell death caused by iron and erastin in CF cells and significantly decreased LDH release.
To investigate the role of CFTR in ferroptosis, iron and erastin were added to a cancer cell line that produces high amounts of CFTR. These cells did not undergo significant cell death by iron and erastin unless treated with a compound that blocked CFTR function. Use of the CFTR inhibitor in healthy cells, after iron and erastin exposure, induced cell death.
“These data suggest that CFTR functions as a potential negative regulator of ferroptosis,” the scientists wrote.
CF cells exposed to iron and erastin demonstrated a three times increase in lipid peroxides — a key feature of ferroptosis. This response was significantly reduced in the presence of DFO or Fer-1. In contrast, healthy cells treated with iron and erastin remained unaffected. A highly toxic byproduct of lipid peroxide degradation called MDA was also higher in CF cells after iron and erastin treatment than in healthy cells.
“These findings suggest that [CF cells] have an increased tendency for lipid peroxidation compared to [healthy] cells,” the scientists added.
As demonstrated in other reports, reduced levels of GSH, which counteracts the effects of free radicals, were found in CF epithelial cells compared with healthy cells. GSH further decreased upon iron and erastin exposure.
Treatment with either DFO or Fer-1 did not affect GSH reduction, “which was expected since the effects of iron chelation (DFO) and inhibition of lipid peroxide formation (Fer-1) are elicited downstream of GSH production,” the researchers wrote.
GPX4 is an enzyme that, with GSH, protects cells against lipid peroxidation and is a modulator of ferroptosis. Although GSH levels were reduced by iron and erastin treatment in both CF and non-CF cells, GPX4 production remained unchanged in healthy cells, whereas it decreased significantly in CF cells.
“These observations suggest that part of the survival propensity of [healthy] cells may be due to an enhanced capacity to continue to prevent lipid hydroperoxides formation, even in the setting of reduced levels of GSH,” the team wrote.
Finally, a type of inflammatory-related cell death called necroptosis appeared to enhance the susceptibility of CF cells to ferroptosis because cell death was partly rescued by necroptosis-blocking compounds. Both cell lines exposed to iron and erastin increased pro-inflammatory signaling proteins; however, the levels were much higher in the CF cells.
“Taken together, our results suggest that FAC and erastin-induced ferroptosis in CF cells shares some common pathways with necroptosis,” the team added. “However, further studies are required to clarify the mechanism of cross-talk between these two pathways in cystic fibrosis.”
“These studies suggest that the increased susceptibility of CF AECs to ferroptosis is linked to abnormal intracellular ferrous iron accumulation and reduced antioxidant defences,” the investigators concluded. “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,” they added.
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