Abstract
Purpose :
Tear levels of CX3CL1/fractalkine, a chemokine implicated in inflammation, were found increased in dry eye patients. Benzalkonium chloride (BAC) is a well-known preservative used at low doses in eyedrops for which toxic effects on ocular surface were described, mainly based on oxidative stress, apoptosis and chronic inflammation mechanisms. BAC not only alters the ocular surface epithelia but also corneal innervation, emerging from the trigeminal ganglion (TG).
Our aim is to investigate the role of CX3CL1/CX3CR1 axis in an in vitro BAC Toxicity-Induced Dry Eye (TIDE) model on a TG cell culture, in relation with the cell death mechanisms induced, apoptosis, ferroptosis, and pyroptosis.
Methods :
We established a reproducible method for primary cultures of TG from wild type (WT) or CX3CR1-KO mice. We investigated the modulation of oxidative stress (CellRox assay, HO1), apoptosis (CASP3), ferroptosis (GPX4) and pyroptosis (NLRP3, GSDM) after exposure to a low concentration (5.10-4%) of BAC. A RT-PCR gene expression kinetic study (1h, 2h, 4h, 6h, 8h and 24h of BAC exposure) and western blot analysis were conducted.
Results :
We found that BAC-induced oxidative stress was reduced at 4h, correlated to a major increase (from 2h to 24h) of HO-1 gene expression in CX3CR1-KO compared to WT. Moreover, GPX4 protein, known to protect against ferroptosis, was depleted at 2h, its gene expression being only upregulated at 1h and 2h in presence of CX3CR1. The increase of CASP3 gene expression was delayed in CX3CR1-KO mice (6h instead of 2h in WT). As for inflammatory cell death processes, NLRP3 inflammasome and GSDM pyroptosis modulator regulations occurred at later time points and were more important in presence of CX3CR1. Indeed, NLRP3 gene expression was upregulated at 24h only in WT whereas GSDM gene expression and protein level were increased at 6h only in WT. Nevertheless, GSDM gene expression at 24h was increased in both WT and CX3CR1-KO mice.
Conclusions :
We confirm that BAC induces not only apoptosis but also other emerging cell death mechanisms with key events occurring in a kinetic manner as follows: 1. oxidative stress, 2. apoptosis, 3. inflammasome activation and pyroptosis. In absence of CX3CR1, oxidative stress is reduced and this effect impacts the subsequent cell death mechanisms. Further studies are needed to deepen the exact role of CX3CR1 axis on cell death mechanisms involved in TIDE.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.