Impact of ergothioneine on idiopathic pulmonary fibrosis markers in human lung epithelial cells in vitro
Authors: Ehrhardt Carsten , Koay Aaron , Clerkin Caoimhe , Selo Mohammed Ali 
Published: June 2021

The pathology of idiopathic pulmonary fibrosis (IPF) remains unknown. Tobacco smoking is a risk factor for IPF and tobacco smoke‐induced oxidative stress can initiate/worsen epithelial‐mesenchymal transition (EMT) in pulmonary epithelial cells, leading to IPF. Ergothioneine (ET) is an antioxidant amino acid available exclusively from the diet. ET requires its physiological transporter OCTN1 (SLC22A4) for cellular uptake and accumulation. Genetic variants of SLC22A4 have been associated with inflammatory bowel diseases and COPD, diseases with fibrotic phenotype. In this study, we tested the hypothesis that ET protects lung epithelial cells in vitro from factors implicated with EMT.NCl‐H441 and A549 cells were cultured in RPMI‐1640 medium supplemented with 5% FBS and 1% sodium pyruvate, at 37°C and in 5% CO2 atmosphere. In some experiments, the medium was supplemented with 250 or 500 μM ET. To assess the impact of tobacco smoke, cells were cultured in the presence of 5 or 10% cigarette smoke extract (CSE) for 24h. CSE was generated by bubbling one Kentucky 3R4F research cigarette through 10ml of medium. The CSE was filtered and diluted before use. To initiate EMT, cells were exposed to 5ng/mL TGF‐β1 in serum‐free medium for 3 days in the respective experiments.Cell proliferation was determined by CyQUANT® NF assay. Cell shape investigation was performed by phase‐contrast microscopy and ImageJ software. A scratch wound assay was carried out to measure wound closure. Before the assay, cells were subjected to serum‐deprivation for 24h. Monolayers were wounded with a pipette tip, followed by 1‐day of serum‐free culture. The wound area was measured by phase‐contrast microscopy using the Cell^A software.The cell proliferation assay showed that CSE‐attenuated cell proliferation rate was significantly reduced by ET in NCl‐H441 cells. However, in A549 cells, their rapid proliferation rate might have compromised the sensitivity of the assay. In A549 cells, decreased circularity and increased aspect ratio were measured after TGF‐β1 treatment. These effects were significantly reduced when cultured in the presence of ET. In the scratch assay, increased cell migration was observed in CSE‐treated NCl‐H441 cells, but the effect was diminished after ET treatment. These data suggest that ET not only protects lung epithelial cells from CSE‐induced cell death, but also prevent CSE‐induced cell migration, thereby ameliorating EMT. Of note, in NCl‐H441 cells, TGF‐β1 failed to establish signs of EMT.Future research will investigate the effect of ET on molecular markers of EMT such as α‐SMA, vimentin and collagen.Support or Funding InformationThis work has been financially supported by a Clinical Investigator Award from the Flight Attendant Medical Research Institute (FAMRI CIA 130016). M.A.S. is the recipient of a PhD bursary from the Iraqi Ministry of Higher Education and Scientific Research (MOHESR). A.K. is the recipient of a Laidlaw Undergraduate Research and Leadership Programme.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.