Abstract
Purpose :
The purpose of this study was to determine the effects of oxidative stress on phagocytosis, barrier function and both redox and innate immune signaling in two in vitro models of human retinal pigment epithelium (RPE) cells, human telomerase reverse transcriptase-overexpressing RPE (hTERT-RPE) and induced pluripotent stem cell-derived RPE (iPSC-RPE) cells.
Methods :
Cells were grown in Dulbecco’s modified Eagle’s medium:F-12 + 10% FBS (with non-essential amino acids, 1% B27 supplement, 10ng/ml triiodothyronine and 100µM taurine for iPSC-RPE only) to full confluence for experiments. Cells were exposed to 50µM or 200µM tert-butyl hydroperoxide (tBHP) in media for 24 hours to induce mild or severe oxidative stress, respectively. Fluorescein isothiocyanate (FITC)-conjugated latex beads of 1µm diameter were used in phagocytosis assays to measure cell function. Actin was labeled with phalloidin-Alexa Fluor 594 and immunocytochemistry was performed to detect zonula occludens 1 (ZO-1), nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) immunoreactivity. Cells were imaged with confocal microscopy (Leica TCS SP5) and image analysis was performed using ImageJ software. Transepithelial electrical resistance (TEER) measurement was conducted with an Ussing chamber system to determine RPE cell barrier function.
Results :
Mild oxidative stress (exposure to 50µM tBHP for 24 hours) had no apparent effect on RPE cell size or morphology as determined by microscopy. Mild oxidative stress also had no effect on phagocytosis, but led to increases in ZO-1, Nrf2 and NFκB immunoreactivity, as well as to a 45% increase in TEER. Higher levels of oxidative stress (exposure to 200µM tBHP for 24 hours) led to changes in cell size and morphology, but did not result in cell death. RPE cells undergoing such severe oxidative stress displayed also impaired phagocytosis, altered and less organized distribution of actin and ZO-1 immunoreactivity and decreased TEER indicative of impaired RPE cell barrier function.
Conclusions :
Oxidative stress induces specific structural and functional changes in cellular signaling in in vitro models of human RPE cells. These changes include an altered cellular redox homeostasis and innate immune signaling potentially contributing to adaptive functional responses in RPE cells.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.