Recombinant human PRG4 reduces pro-inflammatory cytokine release from corneal epithelial cells after endotoxin and poly(I:C) insults

Abstract

Purpose : In addition to its role in joint lubrication, proteoglycan 4 (PRG4; lubricin) has been shown to be produced by the tear-secreting tissues of the eye, where it protects the cornea and conjunctiva against the shearing forces associated with blinking. Furthermore, recombinant human proteoglycan 4 (rhPRG4) is currently in clinical trials for the treatment of moderate dry eye. The purpose of this study was to evaluate the effects on rhPRG4 on pro-inflammatory cytokine release from human corneal epithelial cells after endotoxin (lipopolysaccharide, LPS) and poly(I:C) insult.

Methods : Human corneal epithelial (HCE-T) cells were seeded at a density of 250,000 cells/cm² in a 96-well plate. Cells were grown to approximately 70% confluency for 48 h and treated with either rhPRG4 (300 µg/ml) or saline (0.9% NaCl) vehicle for 24 h. Media was replenished and cells were exposed to endotoxin (lipopolysaccharide, LPS from Salmonella typhimurium, 100 ng/ml), poly(I:C) (10 µg/ml), or saline vehicle in the presence or absence of rhPRG4/saline. The supernatant was collected after 5 h and analyzed by Human Cytokine/Chemokine 48-Plex Discovery Assay® Array (Eve Technologies, Calgary, AB, CA).

Results : LPS and poly(I:C) resulted in release of significant levels of pro-inflammatory cytokines, notably TNFα, IL-6, IL-8, GROα and MCP-1. Though rhPRG4 reduced the release of TNFα (n=4, p<0.01), IL-8 (n=4, p<0.05), GROα (n=4, p<0.001) and MCP-1 (n=4, p<0.01), it did not affect IL-6 secretion (n=4, p>0.05) following LPS insult. Similarly, rhPRG4 decreased secretion of TNFα (n=4, p<0.01), IL-8 (n=4, p<0.01) and GROα (n=4, p<0.01) after poly(I:C) insult. Interestingly, rhPRG4 resulted in a statistically significant reduction of IL-6 (n=4, p<0.05) secretion, but did not prevent MCP-1 (n=4, p>0.05) secretion after poly(I:C) insult.

Conclusions : Recombinant human PRG4 exerts potent anti-inflammatory effects by reducing pro-inflammatory cytokine release from human corneal epithelial cells following multiple stimuli. Our results support future therapeutic development of rhPRG4 for ocular surface and anterior segment disorders associated with increased pro-inflammatory cytokine secretion.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.