Trehalose Protects Human Corneal Epithelial Cells from Inflammation by Promoting Autophagic Flux via TFEB Activation

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 role and potential mechanism of trehalose, which suppresses inflammatory response via autophagy activation 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 of trehalose, for different times (4-48 hours). The mRNA expression by HCECs was determined by reverse transcription and quantitative real time PCR (RT-qPCR). The protein production was evaluated by ELISA, Western blotting and immunofluorescent assays for the cells and their conditioned medium from HCEC cultures.

Results :
The expression of pro-inflammatory cytokines, TNF-α, IL-1β and IL-6, and chemokine IL-8 was significantly stimulated at mRNA and protein levels in HCECs exposed to hyperosmotic medium (450 mOsM). These inflammatory mediators were largely suppressed by prior-treatment with trehalose at 0.5-1.5% while 1.0% showed the best effect. Interestingly, trehalose was found to activate autophagosome formation in HCECs, as evidenced by increased expression and production of autophagy related genes, Beclin 1 and ATG5, as well as the increase of IC3 protein turnover to form IC3-II, as evaluated by RT-qPCR and Western blot analysis. P62 protein, also known as SQSTM1/Sequestome 1, was observed to decrease significantly, indicating that trehalose enhances autophagic flux. Furthermore, the immunofluorescent staining and Western blot showed that the transcription factor EB (TFEB), a master gene for autophagy-lysosome pathway, was activated with translocation from cytoplasm to nucleus by trehalose in HCECs exposed to hyperosmolarity.

Conclusions :
Our findings demonstrate that hyperosmotic stress stimulates the expression and production of proinflammatory mediators in HCECs. Trehalose, not only serving as an osmoprotectant, but also functioning as an autophagy enhancer, suppresses these inflammatory responses by promoting autophagic flux via TFEB activation in HCECs exposed to hyperosmotic stress.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.