Osmolarity-dependently Protective Effects of Trehalose on Inflammatory Markers in Primary Human Corneal Epithelial Cells Exposed to Hyperosmotic Stress

Abstract

Purpose : Hyperosmolarity has been recognized as a pro-inflammatory stress in the pathogenesis of dry eye disease. This study was to explore the protective effects of the disaccharide trehalose on expression and production of pro-inflammatory mediators in primary human corneal epithelial cells (HCECs) exposed to hyperosmotic stress.

Methods : Primary HCECs were established from fresh donor limbal tissue explants. The cultures in iso-osmolar medium (312 mOsM) were switched to hyperosmotic media (450 mOsM) by adding 70 mM NaCl, with or without prior incubation of different concentrations (0.1-5%) of trehalose, alone or in combination of another osmoprotectant, L-carnitine, for different times (4-24 hours). The mRNA expression by HCECs was determined by reverse transcription and quantitative real time PCR. The protein production in the conditioned media from cultures was evaluated by ELISA.

Results : The expression of pro-inflammatory cytokines, TNF-α, IL-1β and IL-6, and chemokine IL-8 was significantly stimulated in HCECs exposed to hyperosmotic medium (450 mOsM). This stimulation was largely suppressed at both mRNA and protein levels by prior-treatment with trehalose at 0.5-1.5% while 1.0% showed the best effect. Higher concentrations (2.0-3.0%) of trehalose reduced the suppressive effects on these pro-inflammatory markers in a concentration-dependent manner, and 5% had no suppressive effect, or was associated with higher expression of these markers. This phenomenon was likely due to the higher osmolarity (500~570 mOsM) generated by 2.0-5.0% of trehalose in the HCEC hyperosmolarity model. Prior treatment with 1.0% trehalose in combination with L-carnitine at 10 mM significantly enhanced the inhibitory effects on these pro-inflammatory markers at mRNA and protein levels, causing 20-50% further down-regulation. Further investigations are going on to explore the differential molecular mechanisms initiated by trehalose and L-carnitine.

Conclusions : Our findings demonstrate that hyperosmotic stress stimulates the expression and production of proinflammatory mediators in HCECs. Trehalose functions as an osmoprotectant, producing an osmolarity-dependent suppression of inflammatory responses. L-carnitine further enhances the protective effects initiated by trehalose, suggesting that the 2 compounds may act by different mechanisms and have synergistic effects.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.