The role of autophagy in the pathogenesis of exposure keratitis

Abstract Incomplete tear film spreading and eyelid closure can cause defective renewal of the ocular surface and air exposure‐induced epithelial keratopathy (EK). In this study, we characterized the role of autophagy in mediating the ocular surface changes leading to EK. Human corneal epithelial cells (HCECs) and C57BL/6 mice were employed as EK models, respectively. Transmission electron microscopy (TEM) evaluated changes in HCECs after air exposure. Each of these models was treated with either an autophagy inhibitor [chloroquine (CQ) or 3‐methyladenine (3‐MA)] or activator [Rapamycin (Rapa)]. Immunohistochemistry assessed autophagy‐related proteins, LC3 and p62 expression levels. Western blotting confirmed the expression levels of the autophagy‐related proteins [Beclin1 and mammalian target of rapamycin (mTOR)], the endoplasmic reticulum (ER) stress‐related proteins (PERK, eIF2α and CHOP) and the PI3K/Akt/mTOR signalling pathway‐related proteins. Real‐time quantitative PCR (qRT‐PCR) determined IL‐1β, IL‐6 and MMP9 gene expression levels. The TUNEL assay detected apoptotic cells. TEM identified autophagic vacuoles in both EK models. Increased LC3 puncta formation and decreased p62 immunofluorescent staining and Western blotting confirmed autophagy induction. CQ treatment increased TUNEL positive staining in HCECs, while Rapa had an opposite effect. Similarly, CQ injection enhanced air exposure‐induced apoptosis and inflammation in the mouse corneal epithelium, which was inhibited by Rapa treatment. Furthermore, the phosphorylation status of PERK and eIF2α and CHOP expression increased in both EK models indicating that ER stress‐induced autophagy promoted cell survival. Taken together, air exposure‐induced autophagy is indispensable for the maintenance of corneal epithelial physiology and cell survival.


| INTRODUC TI ON
The outer layer of the normal cornea is composed of a non-keratinized, non-secretory epithelium covered with a tear film layer. It plays a pivotal role in the maintenance of normal ocular surface functions and refractive properties. 1 Eyelid closure and blinking contribute to maintaining ocular surface integrity since they prevent the corneal surface from desiccation and compromise of epithelial barrier function, which may induce oxidative stress and deprive corneal epithelial cells of essential nutrient support. 2 If air exposure EK develops as a consequence of defective epithelial renewal, incomplete eyelid closure and tear spreading, this pathological condition may lead to chemosis, corneal erosion, melting, infectious keratitis and even corneal perforation. 3,4 The clinical diagnosis of EK is based on patient history and physical examination findings. Patients frequently present with a presumed diagnosis of 'dry eye', but extended corneal air exposure is the underlying aetiology revealed by ophthalmic examination. To unravel the pathophysiology of this condition and other ocular surface diseases, 5,6 Li et al constructed an ex vivo model of air exposure keratopathy, in which human limbal explants were cultured at an air-liquid interface (airlift). 7 Autophagy reutilizes cellular proteins and damaged organelles to derive metabolic energy during starvation or stress. 8,9 This function plays a pivotal role in cell survival in many diseases. 10,11 Cell death can be induced in high autophagy cells by degradative processes such as ischaemia/reperfusion-induced death of cardiac myocytes.
Furthermore, exorbitant autophagy activation can also contribute to pathological changes such as the liver fibrosis and cirrhosis. 12,13 Alternatively, autophagy activation can be beneficial during various pathological and physiological states because this response protect cells from compromise of their function by recycling and degrading damaged or dysfunctional organelles, and meanwhile provides a defensive power against infection. In addition, it plays a salutary role in diabetes, heart failure and neurodegenerative diseases. 14 It is noteworthy that, in some diseases, such as cancer, it is still not fully understood whether autophagy exerts beneficial or detrimental effects. 15,16 Thus, treatment of many human diseases can be facilitated by determining the role of autophagy in their pathophysiology.
Up to now, there is no clarity in the literature confirming the role and the extent of autophagy in the process of EK. In this study, we established both in vitro and in vivo air exposure corneal epithelial keratopathy models to determine the contributory roles of autophagy to this process. The results clarify both the role of autophagy in the survival and death of corneal epithelial cells during air exposure keratopathy and its contribution to the underlying pathophysiology.  The mouse air exposure keratopathy model procedure was performed, as described previously, with some modifications for mice. 17 All procedures with animals were performed under general anaesthesia induced by intraperitoneal injection of ketamine hydrochloride (100 mg/kg) and xylazine hydrochloride (12.5 mg/kg). Eye speculums for mice were used to prevent eyelid closure for either 30 min, 1 h, 2 h or 4 h). Experimental mice were killed 24 hours after the initial exposure, and the corneas were collected for qRT-PCR.

| In Vivo experimental procedures
The other corneal samples were immediately collected from the mice after exposure treatment.

| Cell culture
HCECs, simian virus 40 transformed, were obtained from RIKEN Biosource Center, Tokyo, Japan, and were passaged in DMEM-F12 supplemented with 6% heat-inactivated FBS, bovine insulin (7 μg/ mL), human epidermal growth factor (7 ng/mL), and 1% penicillin and streptomycin. For cell airlift cultures, the HCECs were plated at a density of 1 × 10 5 cells/cm 2 into type I collagen-coated six-well inserts. When the cultured HCECs reached 80% confluency, the medium was replaced with 0.6 mL fresh medium to keep the cells at the air-liquid interface. In some airlift cultures, specific autophagy inhibitors, either CQ (10 μM) or 3-MA (5 mM) was added to the culture medium. Cells were cultured at 37 ℃ in 5% CO 2 and the medium was replaced every 2 days.

| Slit-lamp microscopic observation
Mice were examined under the slit lamp microscope 24 hours after the air exposure treatment. All of the corneal images were taken by an experienced researcher. The corneal epithelial barrier function was detected under cobalt blue light with 0.5% fluorescein sodium eye drops.

| Transmission electron microscopy (TEM)
Cultured cells or corneas were collected and immediately fixed in 2.5% glutaraldehyde in 0.2 M phosphate buffer saline (PBS, pH 7.2) followed by 2% aqueous osmium tetroxide. Subsequently, they were dehydrated using graded ethanol series and then embedded.

| Histological characteristics and immunostaining
Cultured HCECs or mouse corneal frozen sections were hydrated in PBS after fixed in 4% paraformaldehyde for 20 minutes, followed by incubation in 0.2% Triton X-100 for 10 minutes. After rinsing three times with PBS for 5 minutes each and pre-incubation for 1 hour with 2% bovine serum albumin at room temperature to block non-specific staining, they were incubated with primary antibodies (anti-LC3B and SQSTM1/p62) overnight at 4℃. After washing with PBS three times for 10 minutes each, specimens or cells were incubated with secondary antibodies for 1 hour. After washing with PBS three additional times for 15 minutes, sections or cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Dalian Meilun Biotechnology Co., Ltd, Dalian, China) and then mounted for analysis under a Leica DM2500 microscope (Leica Microsystems, Wetzlar, Germany).

| Western blot assay
Cells and corneas were harvested in RIPA buffer (Cell Signaling Technology, Boston, MA, USA). Then they were lysed on ice for 30 minutes, before being centrifuged at 4°C at 14,000 rpm for 10 minutes. After removal of the precipitate, the concentration of extracted proteins was determined with the BCA assay using a commercial kit (Thermo Fisher Scientific, Waltham, MA, USA). Then, the protein samples adjusted to the same concentration were mixed thoroughly with 5 × SDS loading buffer, and heated for 10 minutes at 100°C for denaturation. The protein samples were separated by electrophoresis on 8% or 12% SDS-PAGE gels, and then transferred onto polyvinylidene difluoride membranes (Roche, Indianapolis, IN, USA). After being blocked in 2% BSA for 1 hour, the membranes were incubated with a primary antibody (1:1000) overnight at 4°C. Primary antibodies against LC3B, SQSTM1/p62, Beclin1, CHOP, AKT, p-AKT, mTOR, p-mTOR, PERK and p-PERK were used in this study. On the next day, membranes were rinsed in TBST for 10 minutes thrice, and then incubated with horseradish peroxidase-conjugated goat antirabbit or anti-mouse IgG (Bio-Rad, Hercules, CA, USA) (1:10000) for 1 hour. After three rinsed in TBST, a membrane was analysed in a ChemiDoc XRS imaging system (Bio-Rad). The optical density (OD) was determined using the software of Quantity One. Anti-β-actin mouse monoclonal antibody (1:10000) acted as a loading control.

| In Situ TUNEL assay
To measure end-stage apoptosis, in situ terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) was performed on the corneas or HCECs after air exposure for various durations, according to the DeadEnd Fluorometric TUNEL System protocol. Cells or sections were counterstained in mounting medium with DAPI after TUNEL. The fluorescent dye-conjugated dUTP-labelled DNA and DAPI were visualized under a confocal laser scanning microscope (Fluoview FV1000; Olympus, Tokyo, Japan).

| Statistical analysis
We conducted a one-way ANOVA test to analyse the cell counting data, Western blot and quantitative real-time PCR, followed by a post hoc analysis Tukey test or a Student's t test established significance of differences between the groups. A value of P < 0.05 was considered statistically significant.

| RE SULTS
3.1 | Autophagy activation in air-exposed human corneal epithelial cells ting showed that the ratio of LC3-II/LC3-I was significantly higher in air-lifted cells than that in untreated controls, while the p62 protein level was reduced 24 hours after the air-lift period ( Figure 1E-H).
These findings demonstrate that the air-lift method effectively induces autophagy in HCECs in a time-dependent manner.

| Autophagy improves air-lifted HCECs survival
Autophagy may have different effects on cell survival in different environments. 21 To make such an assessment in HCECs, the individual effects of the autophagy inhibitors, [chloroquine (CQ; 10 μM) or 3-methyladenine (3-MA; 5 mM)] on apoptosis was evaluated in air-lifted HCECs. TUNEL staining was conducted to observe the effect of autophagy on the survival of air-lifted HCECs. In medium submerged HCECs, almost no apoptosis was seen ( Figure 2A); however, in an air-lifted HCECs culture, in the absence of an autophagy inhibitor, apoptosis was evident, although no significant difference was seen between 6 and 24 hours.
Compared with the normal control group, in the presence of the autophagy inhibitors, CQ or 3-MA, the number of apoptotic cells increased which was accompanied by a significant decline in the total cell number ( Figure 2B).

| An air exposure-induced corneal injury mouse model causes autophagy
To determine the effect of air-lifting on autophagy induction in vivo, we developed an air exposure injury mouse model. fluorescence intensity decreased suggesting that autophagy may begin to wane after a 2 hours exposure. Western blot analysis showed that LC3II expression increased at 0.5 hours, and Beclin1 was significantly up-regulated at 1 hour ( Figure 4B and C). These results suggest that autophagy was activated in this air-lifted autophagy mouse cornea model.

| Autophagy improves corneal epithelial cell survival in an air-lifted mouse autophagy model
The aforementioned results suggest that autophagy activation pro-  Figure 6A). Based on these results, the eyes were then isolated in preparation for TUNEL staining. There was no obvious difference in staining between the CQ and solvent groups; however, cell apoptosis increased in the 3-MA group but it decreased in the Rapa group as compared with their solvent vehicle groups ( Figure 6B). Statistical analysis showed that the differences of the number of apoptotic cells between CQ or 3-MA treated group and solvent group were statistically significant ( Figure 6C). In conclusion, Rapa injection activated autophagy and boosted the survival of epithelial cells in this air-lifted autophagy mouse model.

| Air exposure induces endoplasmic reticulum (ER) stress in corneal epithelial cells and activates autophagy via the PI3K/AKT/mTOR signalling pathway
Autophagy (macroautophagy) is induced through both the mechanistic target of rapamycin (mTOR)-dependent autophagy and non-mTOR-dependent autophagy pathways. mTOR regulates the autophagic process by modifying the phosphorylation of Unc-51-like autophagy activating kinase 1 (ULK1), while the activation of mTOR is controlled by the upstream protein kinase B (AKT). 22 To determine the mTOR involvement in air-lift induced autophagy, the individual effects were examined of this stress on mTOR and ULK1 expression F I G U R E 2 Autophagy modulates the apoptosis of HCECs after air exposure. A, Immunofluorescence staining with TUNEL in the HCECs after different treatments, n = 3. B, Quantification of the percentage of TUNEL positive cells. The proportions of TUNEL positive cells in the 6 h and 24 h air-lifted cultured groups were significantly greater than the undamaged controls. Moreover, the numbers of apoptotic cells were significantly increased by CQ and 3-MA, n = 3. **P < 0.01, ***P < 0.001 levels. The results indicate that in this model the mTOR phosphorylation status was inhibited since the p-mTOR content decreased, which is consistent with the increased p-ULK1 content ( Figure 7A and B). These effects were consistent with those in the in vitro autophagy HCECs model suggesting a commonality in upstream signalling control of this process.
The mTOR phosphorylation status is regulated by endoplasmic reticulum (ER) stress in some tissues. In these studies, it was shown that protein kinase R-like endoplasmic reticulum kinase (PERK), CCAAT-enhancer binding protein homologous protein (CHOP) and eukaryotic initiation factor 2α (eIF2α) play important roles in modulating control of mTOR phosphorylation. Therefore, changes in their expression levels serve as markers of mTOR phosphorylation involvement. 23 Similarly, PERK, CHOP and eIF2α in HCECs can serve as readouts of this control and Western blotting showed that they actually increased in a time-dependent manner. At 6 and 24 hours after imposing the air-lift condition, autophagy increased in HCECs as indicated by an increase in LC3 expression and the content of ER stress-related proteins, phosphorylated PERK (p-PERK), CHOP and phosphorylated eIF2α (p-eIF2α) also increased significantly ( Figure 7C). In contrast, following addition of the ER stress inhibitor, salubrinal (sal), the levels of p-PERK and CHOP decreased in conjunction with the level of autophagy, as indicated by a decrease in LC3II expression. These declines occurred since sal inhibited dephosphorylation of eIF2α, which in turn increased levels of p-eIF2α.
It is known that mTOR is involved in the regulation of air-lift injury-induced autophagy ( Figure 7A and B). mTOR is also an important downstream kinase of PI3K/AKT, and together this signalling axis contributes to the regulation of cell proliferation and apoptosis. 24 In the present study, the air exposure condition (6 and 24 h) downregulated the levels of p-mTOR in conjunction with the levels of p-AKT and p-PI3K. However, following the addition of sal to inhibit ER stress, phosphorylation of AKT and PI3K increased ( Figure 7D).
Together, these results imply that air exposure regulates the PI3K/ AKT/mTOR signalling pathway via the induction of ER stress, in turn promoting protective autophagy.

| D ISCUSS I ON
Maintenance of the tear film composition and osmolality is essential for preserving corneal and conjunctival surface health. This requirement is evident since either a change in tear fluid composition ported that autophagy inhibited RGCs apoptosis, using Atg4B -/and Atg5 -/mice. 31 Regarding an association between medium composition and ocular surface health, lacritin, an endogenous tear glycoprotein, reduced oxidative damage to corneal epithelial cells and maintained cellular homoeostasis by inducing autophagy. 32,33 In the present study, we also found that autophagy was rapidly induced subsequent to air-lifted HCECs. Furthermore, following addition of either CQ or 3-MA, autophagy inhibitors, the number of apoptotic cells rapidly increased. These results suggest that in an air-lift model, for achieving these objectives in critically ill patients. The present study found that autophagy may be a potential therapeutic target for air-exposed lesions, proposing a novel avenue for the treatment of ocular surface diseases.
The regulation of autophagy in the treatment of diseases has already achieved good results in various animal models. For instance, the activation of mTOR-dependent autophagy may delay neurodegenerative symptoms in fly and mouse models of Huntington's disease, 35 and a mouse model of Alzheimer's disease. 36 The present study found that with increased exposure time, the levels of p-mTOR gradually decreased, which was negatively correlated with changes in p-ULK1 and LC3II. Therefore, this type of autophagy is considered to be mTOR-dependent. Previously, Rapa has been used as an antiinflammatory drug to treat ocular surface diseases. Shah et al found that Rapa had a significant anti-inflammatory effect in a Sjögren's syndrome mouse model, which inhibited lacrimal gland inflammation and improved ocular surface conditions. 37 Nevertheless, Rapa is also a potent activator of mTOR-dependent autophagy. 38 In our mouse model, following injection of the autophagy activator, Rapa significantly decreased the number of apoptotic cells ( Figure 6) which likely suggests that Rapa promotes ocular surface healing through affecting autophagy in this model.
In recent years, numerous studies have shown that ER stress induces autophagy. Gao et al described ER stress-induced autophagy in a rat cardiac ischaemia-reperfusion model, 39 and Bernales et al reported autophagy involvement in the maintenance of homoeostasis in cells that developed ER stress. 40 Consistently, we found that ER stress appears to be involved in the air-lift model because the ER stress-related proteins, p-PERK, CHOP and p-eIF2α, underwent up-regulation. The autophagy observed in the present study was mTOR-dependent ( Figure 7A and B).
We speculated that the air lift injury-induced ER stress may regulate the initiation of autophagy via the PI3K/AKT/mTOR signalling pathway, since it is an active player in the regulation of cell proliferation and apoptosis. 24 Our hypothesis is supported by our result showing that the ER stress inhibitor, sal ( Figure 7D) activated this pathway. This result is consistent with data reported by Feng and Qin, suggesting that ER stress affects autophagy through the PI3K/AKT/mTOR signalling pathway. 41,42 Other studies showed that glaucoma medications sustained activation of ER stress in corneal epithelial cells. 43   Under extreme stress conditions, an apoptotic signal is triggered, which becomes dominant and leads to cell death. 45 In the present study, air exposure-induced ER stress may have activated autophagy through the PI3K/AKT/mTOR pathway, which plays a positive role in the maintenance of ER homoeostasis and cell survival by degrading and recycling misfolded proteins and damaged organelles. The realization of developing medications that regulate ER stress and autophagy will improve the treatment of ocular surface diseases.
F I G U R E 6 Rapamycin and 3-MA affected both the induction of autophagy and survival in the corneal epithelium after air exposure damage. A, TEM analysis to evaluate the presence of autophagy in mouse corneal epithelium. Norm: normal control group, Vehicle: solvent injection group, 3-MA: 3-MA injection group, Rapa: rapamycin injection group. Autophagosomes are indicated by the red triangle. Nuclei are designated by the letter N, n = 3. B, Immunofluorescence staining with TUNEL in the corneal epithelium after different treatments, C, Quantification of the number of TUNEL positive cells in (B), The number of TUNEL positive cells in Vehicle group was significantly greater than the untreated control. Moreover, the numbers of apoptotic cells were significantly increased by 3-MA and decreased by rapamycin, n = 3. **P < 0.01