Blocking IL-6 trans-signaling mitigates benzalkonium chloride-induced inflammation, oxidative imbalance, ER stress, and barrier disruption in human corneal epithelial cells.
Lax Karina K, Glass Joshua J, Aliste Antonella A, Sharma Ashok A et al.
Benzalkonium chloride (BAC), a commonly used preservative in ophthalmic formulations, elicits a pro-inflammatory cytokine response and cytotoxic effects in corneal epithelial cells, contributing to ocular surface injuries associated with dry eye disease (DED). Interleukin-6 (IL-6) trans-signaling is a well-established pro-inflammatory pathway that contributes to endothelial and epithelial barrier dysfunction. Thus, this study investigated whether targeted inhibition of IL-6 trans-signaling with sgp130Fc could attenuate BAC-induced cytotoxic injury in corneal epithelial cells. Human telomerase-immortalized corneal epithelial (hTCEpi) cells were exposed to 0.001% BAC for 30 min in the presence or absence of 10 μg/mL sgp130Fc. Markers of inflammation, oxidative imbalance, endoplasmic reticulum (ER) stress, and epithelial barrier disruption were assessed using enzyme-linked immunosorbent assay, reverse transcriptase polymerase chain reaction, and western blot analyses. Reactive oxygen species (ROS), antioxidant capacity, and intracellular calcium flux were quantified using colorimetric, fluorescence-based, and luminescence-based assays. Epithelial barrier function was assessed using electric cell-substrate impedance sensing. Statistical analyses were performed using one-way analysis of variance followed by Tukey's or Holm-Šídák multiple-comparison tests, with P < 0.05 considered statistically significant. BAC exposure activated IL-6 trans-signaling as indicated by an increased sIL-6R/sgp130 ratio, reduced mIL-6R levels, and unchanged gp130 levels. BAC also increased secreted IL6 levels, IL6RA expression, and downstream inflammatory mediators MCP1 and ICAM1, all of which were attenuated by sgp130Fc. BAC-induced oxidative imbalance was characterized by increased ROS and hydrogen peroxide production, reduced antioxidant enzymes, and diminished total antioxidant capacity, whereas sgp130Fc mitigated these effects. In addition, BAC increased intracellular calcium levels and ER stress markers, including HSP72, BiP, PERK, and Ero1α, and impaired epithelial barrier function as observed by reduced trans-epithelial electrical resistance and decreased β-catenin, occludin, and claudin-1 levels. sgp130Fc mitigated increases in intracellular calcium, partially alleviated ER stress responses, and preserved epithelial barrier integrity and junctional protein levels. These findings indicate that IL-6 trans-signaling contributes to BAC-induced corneal epithelial injury, suggesting that targeting this pathway may have therapeutic potential for DED and related ocular surface inflammatory disorders.