Autophagy Activation Protects Ocular Surface from Inflammation in a Dry Eye Model In Vitro.

Ding Chen,Stephen C Pflugfelder,Zhao Liu,Ning Gao,Xin Chen,Jinmiao Li,De-Quan Li,Fang Bian

doi:10.3390/ijms21238966

Abstract

Inflammation is the main pathophysiology of dry eye, characterized by tear film instability and hyperosmolarity. The aim of this study was to investigate the association of inflammation and cellular autophagy using an in vitro dry eye model with primary cultured human corneal epithelial cells (HCECs). Primary HCECs cultured with fresh limbal explants from donors were switched to a hyperosmotic medium (450 mOsM) by adding sodium chloride into the culture medium. We observed the stimulated inflammatory mediators, TNF-α, IL-1β, IL-6 and IL-8, as well as the increased expression of autophagy related genes, Ulk1, Beclin1, Atg5 and LC3B, as evaluated by RT-qPCR and ELISA. The immunofluorescent staining of LC3B and Western blotting revealed the activated autophagosome formation and autophagic flux, as evidenced by the increased LC3B autophagic cells with activated Beclin1, Atg5, Atg7 and LC3B proteins, and the decreased levels of P62 protein in HCECs. Interestingly, the autophagy activation was later at 24 h than inflammation induced at 4 h in HCECs exposed to 450 mOsM. Furthermore, application of rapamycin enhanced autophagy activation also reduced the inflammatory mediators and restored cell viability in HCECs exposed to the hyperosmotic medium. Our findings for the first time demonstrate that the autophagy activation is a late phase response to hyperosmotic stress, and is enhanced by rapamycin, which protects HCECs by suppressing inflammation and promoting cells survival, suggesting a new therapeutic potential to treat dry eye diseases.

Highlights

Dry eye is a common disease that affects up to 50% of the population worldwide [1]
We explored a protective role of autophagy activation in suppressing inflammation in dry eye condition using an in vitro model with primary human corneal epithelial cells (HCECs) under hyperosmotic stress
We found the experimental evidence that autophagy activation is a late response to hyperosmotic stress in primary cultured HCECs

Summary

Introduction

Dry eye is a common disease that affects up to 50% of the population worldwide [1]. It is characterized by ocular surface discomfort, tear film instability, increased osmolarity, as well as inflammation and even damage [2,3,4]. As a result of insufficient production [6] and excessive evaporation [7] of tear, hyperosmolarity has been proven as a contributing factor to the inflammatory reaction of the ocular surface in dry eye patients [8,9] and mouse models [10,11,12]. Artificial tears as supplementation and anti-inflammatory therapy are the main treatments for most dry eye patients [18]

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