Oxidative stress in corneal injuries of different origin: Utilization of 3D human corneal epithelial tissue model

Journal Pre-proof Oxidative stress in corneal injuries of different origin: Utilization of 3D human corneal epithelial tissue model Yulia Kaluzhny, Miriam W. Kinuthia, Allison M. Lapointe, Thoa Truong, Mitchell Klausner, Patrick Hayden PII:

S0014-4835(19)30513-5

DOI:

https://doi.org/10.1016/j.exer.2019.107867

Reference:

YEXER 107867

To appear in:

Experimental Eye Research

Received Date: 10 July 2019 Revised Date:

10 October 2019

Accepted Date: 4 November 2019

Please cite this article as: Kaluzhny, Y., Kinuthia, M.W., Lapointe, A.M., Truong, T., Klausner, M., Hayden, P., Oxidative stress in corneal injuries of different origin: Utilization of 3D human corneal epithelial tissue model, Experimental Eye Research (2019), doi: https://doi.org/10.1016/ j.exer.2019.107867. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Oxidative Stress in Corneal Injuries of Different Origin: Utilization of 3D Human Corneal Epithelial Tissue Model.

MatTek Corporation, 200 Homer Avenue, Ashland, MA 01721, USA,

Corresponding author: Yulia Kaluzhny, [email protected], 1-508-881-6771, x229 Miriam W Kinuthia, [email protected] Allison M Lapointe, [email protected] Thoa Truong, [email protected] Mitchell Klausner, [email protected] Patrick Hayden, [email protected]

Declaration of interest All the authors are employees of MatTek Corporation.

1

Abstract The purpose of the current work was to utilize a three dimensional corneal epithelial tissue model to study dry eye disease and oxidative stress related corneal epithelial injuries for the advancement of ocular therapeutics. Air-liquid interface cultures of normal human corneal epithelial cells were used to produce 3D corneal epithelial tissues appropriate for physiologically relevant exposure to environmental factors. Oxidative stress was generated by exposing the tissues to non-toxic doses of ultraviolet radiation (UV), hydrogen peroxide, vesicating agent nitrogen mustard, or to desiccating conditions that stimulated morphological, cellular, and molecular changes relevant to dry eye disease. Corneal specific responses, including barrier function, tissue viability, reactive oxygen species (ROS) accumulation, lipid peroxidation, cytokine release, histology, and gene expression were evaluated. Three dimensional (3D) corneal epithelial tissue model structurally and functionally reproduced key features of molecular responses of various types of oxidative stress-induced ocular damage. The most pronounced effects for different treatments were: UV irradiation - intracellular ROS accumulation; hydrogen peroxide exposure - barrier impairment and IL-8 release; nitrogen mustard exposure - lipid peroxidation and IL-8 release; desiccating conditions - tissue thinning, decline in mucin expression, increased lipid peroxidation and IL-8 release. Utilizing a PCR gene array, we compared effects of corneal epithelial damage on the expression of 84 oxidative stress-responsive genes and found specific molecular responses for each type of the damage. Topical application of lubricant eye drops improved tissue morphology while decreasing lipid peroxidation and IL-8 release from tissues incubated at desiccating conditions. This model is anticipated to be a valuable tool to study molecular mechanisms of corneal epithelial damage and aid in the development of therapies against dry eye disease, oxidative stress- and vesicant-induced ocular injuries. Key words: dry eye disease, oxidative stress, ocular toxicity, ocular injury, chemical and biological weapons, alternative to animal testing, 3D tissue model; 3D-HCE, 3D tissue model Abbreviations: 3D-HCE, three dimensional human corneal epithelial tissue model, UV, ultraviolet radiation; OS, oxidative stress; ROS, reactive oxygen species; DED, dry eye disease; TEER, 2

transepithelial electrical resistance; TEM, transmission electron microscopy; SCC, standard culture conditions; DSC, desiccating stress conditions; NC, negative control; NM, nitrogen mustard; HP, hydrogen peroxide

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Introduction The cornea is an exposed mucosal surface of the eye that plays a crucial role as a barrier against many types of corneal injury, trauma, and infection. The corneal epithelium is a stratified, nonkeratinized squamous epithelial tissue containing 5 to 7 cell layers and is directly involved in protecting cornea from UV irradiation, harmful pollutants and injury (Bazeer et al., 2019; Cejka and Cejkova, 2015; Nakamura et al., 2007; Nelson et al., 2017). The healthy corneal epithelial tissue was shown to be rich in antioxidant enzymes involved in the removal of free radicals and reactive oxygen species (ROS). Corneal aldehyde dehydrogenase (ALDH) is a major soluble protein of the mammalian cornea with important role in detoxification of cytotoxic aldehydes that are generated in cells after membrane lipid peroxidation (Koppaka et al., 2016). Since the eye is continuously exposed to various oxidative conditions, including ambient oxygen, photo-oxidation, ionizing radiation, cigarette smoke, and various forms of pollutants, it is vulnerable to its injurious actions. Oxidative stress (OS) can affect all molecules within the cell and it can also alter enzyme activity, inhibit nucleic acid and protein synthesis, increase the expression of proinflammatory genes and activate the inflammation process which often exacerbates the OS, creating a self-perpetuating, vicious cycle of oxidation and inflammation (Batliwala et al., 2017). OS has been associated with many ocular disorders, including Dry eye disease (DED), age-related macular degeneration, cataracts, diabetic retinopathy, keratoconus, Fuchs’ endothelial dystrophy, bullous keratopathy, and glaucoma. OS also plays a role in corneal injuries due to ultraviolet (UV) irradiation, chemical burns, and exposure to vesicating agents (Cejka et al., 2017; Goswami et al., 2016a; PinazoDuran et al., 2014). In this study, we utilized an in vitro reconstructed 3D human corneal epithelial (3DHCE) tissue model to study the corneal epithelial response to various oxidizing conditions, including DED induced by desiccating conditions, exposure to UV irradiation and chemical agents. Dry eye is a multifactorial disease of the ocular surface and results in discomfort and visual disturbance. DED can involve lachrymal and (or) meibomian glands, corneal innervations, and immune components; it can destabilize the tear film and lead to ocular surface damage and result in aberrations 4

that make performance of everyday tasks difficult and painful (Bron et al., 2017; Lopez-Miguel et al., 2014; Messmer, 2015; Shimazaki, 2018). In the 21st century, the prevalence of DED has increased due to the general aging of the population and the use of certain drugs. Low humidity and dry air, which are the results of modern living where forced air ventilation systems are used for heating and cooling, are also considered as contributing factors to the rise in DED. Dry eye is one of the most common problems of patients seeking ophthalmological care and affects ~30% of those older than 40 years (Nelson et al., 2017; Pflugfelder, 2008; Shimazaki, 2018). Eye trauma accounts for approximately 3% of all visits to the emergency department with the vast majority of these presentations involving corneal injury. Exposure-related burns of the eye can be categorized into chemical burns, radiation burns from ultraviolet sources, and thermal burns. Alkali corneal injuries are the most common due to the prevalence of household cleaning agents containing ammonia and peroxides (Willmann and Melanson, 2019). Another type of chemical exposure that can result in serious eye damage can be caused by vesicating mustard agents. Vesicating agents are potent chemical warfare agents that primarily cause damage to the skin, respiratory organs, and eyes; with eyes being the most sensitive organ to the contact (Goswami et al., 2016a; Kadar et al., 2009). Vesicating agents are reported to cause clinical and histopathological changes related to inflammation and vesication in the eye. These effects are the result of alkylation of major molecules, oxidative stress, lipid peroxidation, and induction of inflammatory responses (Goswami et al., 2016a; Goswami et al., 2016b; Kadar et al., 2009). In the worst cases, chemical damage to the eye can result in blindness. According to the World Health Organization (WHO), corneal blindness is the 4th largest cause of blindness worldwide where either loss of transparency or distortion of the refractive surface causes vision impairment (Flaxman et al., 2017; WHO, 2019). To understand the mechanisms leading to DED and exposure-related corneal injuries, various experimental cell culture and animal models have been developed.

Primary cultures of corneal

epithelial cells or cell lines have been studied under pathological conditions to evaluate cell viability, apoptosis and signaling pathways (Chen et al., 2008; Shivakumar et al., 2018; Yin et al., 2018). However, the main problem with monolayer cell cultures is their inability to simulate physiologically 5

relevant exposure to environmental factors to induce relevant oxidative injury. Several DED and vesicating injury animal models have been developed with rabbit models being the most popular (Barabino and Dana, 2004; Bron et al., 2017; Lee et al., 2019; Nakamura et al., 2007). However, animal models don’t allow study of the cellular and molecular aspects of the exposure or disease in a dynamic fashion, are labor-intensive, limited by species differences, and have ethical concerns. A non-animal, in vivo-like in vitro reconstructed tissue model that mimics human DED and realistic exposures to a variety of harmful conditions is an extremely useful tool to investigate the multiple factors implicated in the ocular surface damage and corneal OS. Emerging technologies such as organotypic tissue models provide opportunities to modernize model systems related to ocular pharmacology and to complement or replace traditional, animal-based experiments. The use of in vitro 3D models can simplify the characterization of specific biological processes that are otherwise difficult to study in vivo (Gonzalez-Andrades et al., 2016; Proulx, 2010; Zaniolo et al., 2013). Several researchers have shown that in vitro models are helpful as tools for toxicity and eye irritation testing (Kaluzhny et al., 2011; Verstraelen and Van Rompay, 2018), permeability and drug transport studies (Toropainen, 2001), and to study mechanisms of ocular surface damage (Meloni et al., 2011). However, the advantages presented by human cell lines or primary nonhuman cells are often overshadowed by incongruous responses and/or gene expression making them non-reliable or irrelevant (Hughes et al., 2007; Whitwell et al., 2015). On the other hand, new methods to isolate and expand primary cells now provide a dependable source of normal human corneal epithelial (NHCE) cells (as opposed to genetically modified cell lines) for the development of novel 3D corneal tissue models (Kaluzhny et al., 2018; Postnikoff et al., 2014; Proulx, 2010). Primary NHCE cells cultured under in vivo-like conditions at the air-liquid interface can address questions concerning mechanisms of ocular damage and avoid species extrapolation and the impact of modified gene expression due to cell immortalization (Ghezzi et al., 2015). The development of in vitro reconstructed human corneal tissues will meet an important, growing need in recreating various aspects of DED and other ocular surface damage conditions and will facilitate the development of ophthalmic medications and antioxidant therapies (O'Neil et al., 2019; Toropainen, 2001; Xiang et al., 2009). 6

The goal of these studies was to investigate an application of in vitro reconstructed 3D normal human corneal epithelial (3D-HCE) tissue model, EpiCorneal, to reproduce morphological, cellular, and molecular changes relevant to DED and various oxidative stress injuries in the in vivo human corneal epithelium. 1. Methods 1.1. In Vitro Reconstructed 3D-HCE Tissue Model The 3D-HCE tissues were cultured using primary normal human donor cells as previously described (Kaluzhny et al., 2018). Human donor corneas were harvested within 24 hours post mortem and shipped by the National Disease Research Interchange (NDRI, Philadelphia, PA) to MatTek in Optisol-GS medium (Bausch & Lomb, Rochester, NY) at 4°C. 3D tissue constructs were grown in microporous membrane cell culture inserts using serum-free medium COR-100-GM (MatTek, Ashland, MA) at standard culture conditions (SCC, 37±1°C, 95±3% relative humidity, and 5±0.5% CO2). The tissues were cultured under submerged conditions with medium contacting both the apical and basolateral sides of the tissue until the cells reached confluence. Once the cells reached confluence, the apical medium was removed thereby exposing the cultures to the air–liquid interface (Fig. 1A and B) which stimulates tissue maturation. All cell strains used for tissue production were screened for mycoplasma, yeast, fungi, bacteria, HIV, and hepatitis B and C; cells from different donors were not pooled together. 1.2. Light Microscopy Light microscopy was utilized to image histology cross-sections of the 3D-HCE tissues as previously described (Fig. 1C and D) (Kaluzhny et al., 2018). 3D-HCE tissues were harvested on day 10 or as indicated. Human donor corneas were cut into wedges, fixed in 10% neutral-buffered formalin and processed in the same way. 1.3. Immunofluorescent Analysis Immunofluorescent imaging of the 3D-HCE tissues was utilized methods similar to those previously reported (Kaluzhny et al., 2018). 3D-HCE tissue constructs were harvested on day 10 or as 7

indicated, fixed and stained with the following antibodies: tight junction sampler pack (ZO-1 [1:20], Occludin [1:20], and Claudin-1 [1:20]), MUC1 [1:50], and Texas Red (all ThermoFisher Scientific, Waltham, MA); Cytokeratins CK3/12 [1:50], Aldehyde Dehydrogenase-3 (ALDH3A1) [1:20], Alexa Fluor-488 [1:400] and Alexa Fouor-555 [1:400] (all Abcam, Cambridge, MA); DAPI and Propidium Iodine (PI) (both Sigma-Aldrich, Burlington, MA). The samples were analyzed by confocal microscopy using an Olympus FV1000 with FluoView™ imaging software (Fig. 2 and 3). 1.4. Transepithelial Electrical Resistance (TEER) Measurements TEER is a sensitive indicator of the 3D tissue barrier integrity. TEER was measured using the EVOM epithelial voltohmmeter and the EndOhm-12 chamber (World Precision, Sarasota, FL, USA) as previously reported (Kaluzhny et al., 2018). The tissues matured to form in vivo-like tissue barrier on day 10 (Fig. 4). 3D-HCE tissues with TEER values over 600 Ω•cm2 were used for the DED model, oxidative stress, and induced corneal injury studies (Kusano et al., 2010; Uematsu et al., 2016). 1.5. Dry Eye Disease (DED) Model 3D-HCE tissues were packaged and stored overnight at 2-8°C to simulate shipping conditions. On the following day, the tissues were equilibrated for 1 hour in the maintenance medium COR-100MM (MatTek Corporation) at SCC (37°C, 95% RH, and 5% CO2) prior to generation of the DED model. The DED model was generated by placing 3D-HCE tissues for up to 72 hours under desiccating stress conditions (DSC) that were characterized by elevated temperatures and lowered humidity when compared to SCC (Barabino et al., 2017; Meloni et al., 2011). The effect of temperature (37°C and 38°C) and humidity (60% to 95% RH) on 3D-HCE tissues was explored. Over-the-counter lubricating eye drops (GenTeal, Alcon) were applied topically (25 µl/tissue) prior to the exposure to DSC and reapplied every 24 hours. 1.6. UV irradiation Following overnight storage as above, the 3D-HCE tissues were equilibrated overnight in COR100-MM medium (MatTek Corporation) at SCC. On the next day, 3D-HCE tissues were transferred into 6-well plates containing PBS (1 ml/well) and irradiated through the lid of the plate. A Hönle SOL500 8

solar simulator was used to deliver full spectrum simulated solar light using h2 filter (Hönle). Doses equivalent of 60 mJ/cm2 and 120 mJ/cm2 of UVB were used (Zheng et al., 2013). Control tissues were incubated at room temperature in the dark for an equivalent period of time. After irradiation, the tissues were returned to COR-100-MM medium and incubated at SCC for 2 or 24 hours prior to analysis. 1.7. Hydrogen peroxide and nitrogen-mustard exposures Following overnight storage, the tissues were equilibrated overnight in COR-100-MM (MatTek Corporation) at SCC. The next day, 300 µl of hydrogen peroxide (HP, Sigma-Aldrich, Burlington, MA) solutions (20 and 50 mM) were topically applied to the 3D-HCE tissues (Fig. 1A). After 2 hours of exposure to HP solution at SCC, the 3D-HCE tissues were rinsed in PBS and analyzed immediately, or post-incubated in fresh medium for 24 hours at SCC prior to analysis. 25 µl of nitrogen-mustard (NM, CAS# 55-86-7, Sigma-Aldrich) solution (7.8 mM) was applied topically to the 3D-HCE tissues as above and incubated for 10 min or 30 min at SCC. After the exposure to NM solution, the 3D-HCE tissues were rinsed in PBS and post-incubated in fresh medium for 2 hours and 24 hours at SCC prior to analysis. 1.8. Tissue viability assay (MTT assay) Tissue viability was analyzed using the colorimetric MTT assay as previously reported (Kaluzhny et al., 2018). Briefly, following the experimental treatments, the 3D-HCE tissues were rinsed and incubated in MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 3 hours. Metabolically active cells convert the MTT reagent to a purple formazan which was extracted and quantified spectrophotometrically at 575 nm. Tissue viability was determined from the ratio of the treated to negative control tissues: %Viability = A

sample

/ANCX100, where: A

sample

– the absorbance of

the extracted solution from the treated tissues; ANC – the absorbance of the extracted solution from the tissues treated with placebo formulations (negative control). The MTT assay is regularly utilized for quality control purposes for MatTek’s in vitro reconstructed tissue models and to determine tissue viability following experimental treatments

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(Kaluzhny et al., 2011). All batches of tissues used in the studies reported herein had an optical density (OD) >1.0. 1.9. Cytotoxicity assay (LDH assay) Lactate dehydrogenase (LDH) is a cytosolic enzyme that if released into the medium, is an indicator of cellular toxicity. LDH release by the 3D-HCE tissues was analyzed using the LDH Cytotoxicity Detection Kit according to manufacturer’s instructions (Clontech/Takara Bio, Shiga, Japan). 3D-HCE tissues treated with 0.1% Triton X-100 for 2 hours were used as the positive control and untreated 3D-HCE tissues were used as negative control. Medium samples were stored at 2-8°C until analysis. 1.10.

Intracellular reactive oxygen species (ROS) accumulation

3D-HCE tissues were packaged and stored overnight at 2-8°C. On the following day, the tissues were equilibrated for 1 hour in COR-100-MM medium and then transferred into fresh COR-100-MM containing 100 µM of DCFH-DA (Sigma) and incubated for 1 hour at SCC. After the 1 hour incubation in DCFH-DA, the 3D-HCE tissues were ready for experimental treatment (UV irradiation or HP and NM exposures). At the end of the treatment, the tissues were rinsed in PBS and lysed in 1% Triton in PBS. The amount of ROS in the samples was quantified using a fluorescent plate reader (Synergy HT BioTek) and 485/520 nm excitation and emission filters. 1.11.

8-Isoprostane and IL-8 release

8-Isoprostane (8-ISO) or Interleukin-8 (IL-8) release into the medium was determined by collecting the COR-100-MM medium 2 hours and 24 hours post-incubation, following tissue treatment (DED, UV irradiation, HP and NM exposures).

The medium samples were stored at -80°C until

analysis. The 8-Isoprostane competitive EIA assay kit (Cayman Chemical, Ann Arbor, MI, USA) and the human IL-8/CXCL8 quantikine ELISA kit (R&D Systems, Minneapolis, MN) were used according to the manufacturers’ instructions. 1.12.

RNA extraction and Real-Time Quantitative Polymerase Chain Reactions (qPCR) 10

Samples for gene expression analysis were obtained from: a) 3D-HCE tissue constructs harvested on day 11 or at the end of the indicated experiment or b) donor corneoscleral buttons obtained from NDRI (Philadelphia, PA). Corneoscleral buttons were excised, incubated with Dispase, and the corneal epithelium was carefully peeled away before it was processed for RNA extraction and cDNA synthesis, as previously described (Kaluzhny et al., 2018). cDNA was evaluated using the SuperArray RT2 qPCR Master Mix and the specific Qiagen primers: MUC1 (Cat. #PPH01085A), MUC4 (Cat. #PPH24163F), MUC16 (Cat. #PPH60570A), ALDH1A1 (Cat. #PPH01723B), ALDH3A1 (Cat. #PPH07009A), TXNRD1 (Cat. #PPH02104F), UGT1A1 (Cat. #PPH01593A), CYP2D6 (Cat. #PPH01259B), CYP2C19 (Cat. #PPH01241C), GAPDH (Cat. #PPH00150F). qPCR was performed as previously described (Kaluzhny et al., 2018). All data were derived from at least three independent measurements and were normalized to the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA. The average threshold cycle Ct, relative ∆Ct (∆Ct = Ct gene of interest – Ct GAPDH)

and ∆Ct expression (2-∆Ct) were determined (Livak and Schmittgen, 2001). Based on the ∆Ct

expression all genes were assigned into one of the following groups: (1) very strong expression (1.0 ≤ 2-∆Ct); (2) strong expression (0.1 ≤ 2-∆Ct < 1.0); (3) moderate expression (0.01 ≤ 2-∆Ct < 0.1); (4) low expression (0.001≤ 2-∆Ct < 0.01); (5) very low expression (0.0001 ≤ 2-∆Ct < 0.001). If Ct > 35, gene expression was considered to be not detected. RT2 Profiler™ Human Oxidative Stress Plus PCR Array (PAHS-065Y) and Human Drug Metabolism (PAHS-002Z) PCR arrays were obtained from SABiosciences.

Gene analysis was

performed using these arrays with a BioRad CFX96 PCR system (BioRad, CA), following the manufacturer’s instructions. 84 genes related to oxidative stress were analyzed. Housekeeping genes GAPDH and beta actin from the gene array were used for gene expression normalization. The average threshold cycle Ct, relative ∆Ct (∆Ct = Ct

gene of interest

– Ct

GAPDH),

and ∆Ct expression (2-∆Ct) were

determined (Livak and Schmittgen, 2001). The fold increase/decrease in expression of a target gene versus a negative control was determined using the following equation: Fold difference = 2-∆(∆Ct), where ∆(∆Ct)= ∆Ct(Experimental) - ∆Ct(Negative Control). 1.13.

Statistical Analysis 11

Results for barrier integrity (TEER), viability (MTT) and cytotoxicity (LDH) assays, intracellular ROS accumulation, 8-Isoprostane and IL-8 release are shown as averages ± standard deviation. Statistical differences were determined using student’s paired t-test. Simple linear regression (R2) was used to establish a correlation coefficient for gene expression between the treated 3D-HCE tissues (exposed to DSC, UVB, hydrogen peroxide or nitrogen mustard) versus the negative control 3D-HCE tissues. 2. Results 2.1. Reconstructed 3D-HCE Tissue Morphology, Ultrastructure and Barrier Function The in vitro reconstructed human 3D-HCE tissues are fully differentiated, well organized corneal epithelial tissue models with morphology similar to native human cornea epithelium (Figures 1C and 1D). Due to the air-liquid interface culture method, the 3D-HCE tissues develop stratified epithelial structure similar to in vivo tissue consisting of: a) single layer of columnar basal cells, b) two to three layers of polygonal wing cells, and c) two to three layers of squamous cells with flattened nuclei. Overall the tissues contain 4-7 layers of cells with average tissue thickness of 50 - 70 µm. The corneal epithelial cells in 3D-HCE reconstructed tissue model expressed corneal epithelial cytokeratins CK3/12 (Fig. 2A and B) and corneal-specific aldehyde dehydrogenase 3A1 (ALDH3A1, Fig. 2C and D).

Immunohistochemical analysis confirmed expression of CK3/12 mainly in the

superbasal and apical layers, with slightly lower expression in the basal layer (Fig 2A and 2B), which suggests that 3D-HCE construct is analogous to the epithelium of an adult corneal button (Merjava et al., 2011). ALDH3A1, known to be important for detoxification of UV-induced oxidative stress, was expressed in the majority of cells located throughout the tissue section (Fig. 2C and D) (Forest et al., 2015; Koppaka et al., 2016). Tight junction proteins: ZO-1 (Fig. 3A and B), occludin (Fig.3C and D) and claudin-1 (Fig. 3E and F), were detected in the apical layers of the 3D-HCE tissue construct (Forest et al., 2015). Since the 3D tissue is a multilayer structure, tight junction proteins and cellular nuclei observed in the images are not in the same focal plane.

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Transmission electron microscopy verified an in vivo-like organization of 3D-HCE epithelium: the outermost cell layers were flat and displayed microvilli, desmosomes, and tight junctions (previously published in (Kaluzhny et al., 2018). The presence of tight junctions is very important for tight barrier development. When the in vitro reconstructed 3D-HCE tissues are cultured at the air–liquid interface, they are able to develop an in vivo–like tight tissue barrier which was confirmed by TEER (Fig. 4). Under-developed 3D-HCE tissues (up to day 8) did not possess tight junctions or a mature barrier and display TEER under 300 Ω·cm2 however, after 10 days in culture, TEER values regularly exceeded 900 Ω·cm2. This coincides with the flattening of apical layers and the development of tight junctions (Fig. 1C and 3), which imparts a tight, in vivo–like tissue barrier. The barrier properties are maintained until at least day 15 by culturing the tissues in COR-100-MM medium (Fig. 4). Experiments using the 3DHCE tissues exposed to various oxidative stress conditions were performed on day 11 when TEER measurements averaged 900 Ω*cm2± 173. Comparable values of TEER were recorded in rabbit and in human studies with intraocular electrodes (Kusano et al., 2010; Uematsu et al., 2016). 2.2. Expression of Tissue Specific Mucins and Detoxification Enzymes by 3D-HCE Tissue Model This study focused on the analysis of gene expression of key corneal cell surface associated mucins (MUC1, MUC4, and MUC16), enzymes involved in detoxification of UV-induced lipid peroxidation and oxidative stress (ALDH1A1, ALDH3A1, TXNED1, and UCT1A1), as well as Phase II detoxifying enzymes known to be important in protection from oxidative stress (glutathione Stransferase,

glutathione

peroxidase,

metallothionein

2A,

pyruvate

kinase,

and

catechol-O-

methyltransferase) (Table 1) (Ablamowicz and Nichols, 2016; Kolln and Reichl, 2016; Koppaka et al., 2016).

Extensive evaluation of gene expression of Phase I and Phase II drug-metabolizing enzymes

and drug transporters by the corneal tissue model and native corneal epithelium has been previously published (Kaluzhny et al., 2018). Mucins expressed on the ocular surface play a vital role in protecting sight. The surface mucins that were investigated, MUC1, MUC4, and MUC16, were expressed in 3D-HCE tissues and the degree 13

of expression was similar for the reconstructed tissues and excised human corneal epithelium (Table 1). Specifically, low to moderate gene expression of the mucin proteins, MUC16 and MUC1, was observed in the excised corneal epithelium and 3D-HCE tissues (0.001 ≤ 2-∆Ct < 0.1) while MUC4 gene expression was very low (0.0001 ≤ 2-∆Ct < 0.001) in both (Table 1). Both aldehyde dehydrogenases (ALDH1A1, ALDH3A1) and the oxidative scavenger, thioredoxin reductase 1 (TXNRD1), were highly expressed in the corneum epithelium and the 3D reconstructed tissues (moderate to very strong expression, Table 1), indicating that the 3D-HCE tissue model is well equipped with enzymes necessary to protect corneal tissue against UV-induced oxidative damage (Koppaka et al., 2016; Wojcik et al., 2013). Phase II drug-metabolizing enzyme UDPglucuronosyltransferase (UGT1A1) gene expression was detected, but at very low and low levels in the excised corneal tissue (0.0001 ≤ 2-∆Ct < 0.001) and the in vitro reconstructed 3D-HCE tissue model (0.001 ≤ 2-∆Ct < 0.01) (Table 1).

In addition to aldehyde dehydrogenases, 3D-HCE tissues showed

robust expression of other Phase II detoxifying enzymes implicated in protection from oxidative stress: glutathione S-transferases (moderate to strong, 0.01 ≤ 2-∆Ct < 1.0), glutathione peroxidases (moderate to very strong, 0.01 ≤ 2-∆Ct) with an exception of GPX3 which had low expression in both the corneal epithelium and 3D-HCE tissues (0.001≤ 2-∆Ct < 0.01). Microsomal glutathione S-transferases had strong to very strong expression (0.1 ≤ 2-∆Ct), metallothionein and pyruvate kinase - very strong (1.0 ≤ 2-∆Ct), and cetechol-O-methyltransferase – strong (0.1 ≤ 2-∆Ct < 1.0) (Table 1). In summary, expression of the corneal specific mucin proteins, oxidative scavenger enzymes, and other Phase II detoxifying enzymes involved in protection from oxidative stress resembled that observed in the epithelial layer of a normal human cornea. These results suggest that the 3D-HCE reconstructed corneal tissue model is able to replicate the main functions of corneal epithelium as they relate to oxidative damages (Table 1). 2.3. In Vitro Dry Eye Disease (DED) Epithelial Model To reproduce corneal epithelial effects of DED in vitro, the 3D-HCE tissues were subjected to the desiccating stress conditions (DSC). The severity of DED was controlled by varying temperatures, 14

relative humidity (RH), and the duration of the incubation at DSC. Under standard culture conditions (SCC, 37°C, 95%RH, and 5% CO2), tissue barrier and morphology could be maintained for up to 16 days (Fig. 4). Since DED and exposure to DSC were shown to lead to disruption of corneal epithelial barrier and increased secretion of inflammatory cytokines (Yamaguchi, 2018), we investigated its effects on barrier integrity, tissue viability, and the release of pro-inflammatory cytokine interleukin 8 (IL8) in the in vitro reconstructed corneal tissues maintained at DSC for 24 to 72 hours. 3D tissues that were incubated at SCC for the same duration were used as negative control (NC) and the parameters obtained from the NC tissues were set as 100%. By decreasing RH to 80% and maintaining the temperature at 37°C, we developed a mild DED in vitro epithelial model, DSC1 (37°C, 80%RH). At DSC1, there was no biologically significant effect on the tissue’s barrier, viability, or IL-8 release; however, significant time-depended lipid peroxidation was observed (Fig. 5). Lipid peroxidation was monitored by measuring the accumulation of prostaglandin 8 iso-PGF2α (8-Isoprostane, 8-ISO) in the culture medium (Fig. 5D). Isoprostanes are a complex family of compounds produced from arachidonic acid via a free-radical-catalyzed mechanism that can be used as reliable markers of lipid peroxidation (Cejkova and Cejka, 2015; Milne et al., 2015; Samar, 2007). Exposure to DSC1 led to significant lipid peroxidation: 2.6, 3.6, and 4.8-fold increases in 8-ISO were observed at 24, 48 and 72 hours, respectively, when compared to the tissues incubated at SCC. For reference, control 3D-HCE tissues incubated in 5 ml of COR-100-MM medium at SCC for 24 hours accumulated 327±38.8 pg/ml of 8-ISO. By decreasing relative humidity to 60% and maintaining the temperature at 37°C, we developed a moderate DED in vitro epithelial model, DSC2 (37°C, 60%RH). Incubation of the in vitro corneal tissues at DSC2 induced a significant impairment of the barrier after 24, 48, and 72 hours of incubation: 74.8±10.8%, 72.2±2.6% and 33.1±10.6%, respectively relative to the 3D tissues incubated at SCC (Fig. 5A). In addition, incubation at DSC2 led to slight, but significant reductions in tissue viability following 48 and 72 hours of incubation: 89.7±2.1% and 89.1±5.4%, respectively, as evaluated by changes in mitochondrial activity of viable cells (MTT assay) (Fig. 5B). It also led to increases in tissue cytotoxicity 15

as assessed by LDH release into the medium following 72 hour of incubation (83.1±5.5%)(Fig. 5C). Furthermore, a significant amount of 8-ISO accumulated in the medium; the highest amount was observed after 72 hours of incubation (16.5-fold increase) (Fig. 5D). DSC2 led to time-dependent secretion of IL-8, the highest amount was also registered after 72 hours (2.6-fold, Fig. 5E).

For

reference, control 3D-HCE tissues incubated at SCC in 5 ml of COR-100-MM medium for 24 and 72 hours released 18.4±5.6 and 56.3±18.5 pg/ml of IL-8, respectively. By decreasing relative humidity to 60% and increasing the temperature to 38°C, we developed a severe in vitro DED epithelial model, DSC3 (38°C, 60%RH). DSC3 led to faster development of the DED phenotype with more severe markers of the disease: highly dehydrated tissues with significantly weakened barrier (Fig. 5A and 6F). Since DSC were developed to mimic physiologically relevant in vivo-like DED conditions without being excessively harmful, 3D-HCE tissues were incubated at DSC3 only up to 48 hours. Tissues barrier declined to 69.1±6.8% and 51.3±5.1% after 24 and 48 hours of incubation (Fig. 5A), which was consistent with progressive tissue dehydration and thinning, reduction of viable layers, and development of cornified cell layers (Fig. 6F) (Glasgow et al., 2010). Tissue thinning was accompanied by only minimal decline in tissue viability (Fig. 5C). However, it led to rapid accumulation of 8-ISO in the medium: increases of 10.6 and 21.3-fold after 24 and 48 hours of incubation when compared to SCC (Fig. 5D). In addition, incubation at DSC3 led to significant, timedependent secretion of IL-8: 3.3 and 5.1-fold increase after 24 and 48 hours of incubation (Fig. 5E). Topical application of eye drops (25 µl/tissue, GenTeal, Alcon) prior and during incubation at DSC3 counterbalanced the effect of the severe DED development by improving tissue morphology and mucin expression (Fig. 6F and G), barrier function (TEER, Fig. 6A), viability (Fig. 6B and C), and 8-ISO and IL-8 release (Fig. 6D and E). Slight decrease of mucin staining of 3D-HCE tissues was also observed following 48 hours incubation at SCC which is expected owing to mucin glycosylation that could negatively affect antibody staining (Ablamowicz and Nichols, 2016). 2.4. Oxidative Stress-Induced Corneal Injury: Response to UV Irradiation, Hydrogen Peroxide, and Nitrogen-Mustard exposure 16

Oxidative stress (OS) plays an important role not only in dry eye disease, but also in corneal injuries such as those caused by irradiation, chemical burns, and exposure to vesicating agents (Cejka et al., 2017; Cejkova and Cejka, 2015; Goswami et al., 2016a; Yin et al., 2018). OS was generated by exposing 3D-HCE tissues to full spectrum solar irradiation equivalent to 60 mJ/cm2 and 120 mJ/cm2 of UVB, or by incubating 3D-HCE tissues with topically applied 20 mM and 50 mM hydrogen peroxide solution (HP) for 2 hours, or 7.8 mM nitrogen mustard solution (NM) for 10 and 30 min. All the treatments were chosen to induce physiologically relevant OS and injury and to avoid extensive tissue damage by necrosis. Barrier function (TEER), tissue viability (MTT assay), cell cytotoxicity (LDH release), lipid peroxidation (8-ISO), cytokine release (IL-8), and tissue morphology (H&E stained-cross sections) were evaluated 2 and 24 hours post-exposure (Fig. 7). Reactive oxygen species (ROS) accumulation was evaluated after 2 hours of post-exposure to avoid a decay of the fluorescent probe; and gene expression was evaluated 24 hours post-exposure to allow for the development of the toxic effect. The most pronounced effect in response to UVB irradiation was accumulation of ROS in the 3DHCE tissues exposed at both, 60 mJ/cm2 and 120 mJ/cm2: 14.9- and 17.0-fold higher than in control tissues that were protected from light (Fig. 7D).

Accumulation of ROS led to elevation of lipid

peroxidation and the release of pro-inflammatory cytokine, IL-8 (Fig. 7E and F). However, no effect on tissue barrier and viability was observed (Fig. 7A-C) that was confirmed by intact tissue morphology following 2 and 24 hours of post-exposure incubation (Fig. 7G1 and 7G2). The most pronounced effect in response to HP treatment was barrier impairment in tissues exposed to 50 mM HP: 64.2±14.2% and 29.9±12.2% immediately after 2-hour treatment and following 24 hours of post-incubation when compared to non-treated tissues (Fig. 7A). The increase in barrier permeability corresponded to a decline in tissue viability, significant lipid peroxidation, and IL-8 release immediately after the 2-hour treatment and following 24-hour post-incubation (Fig. 7).

Relatively

modest increases in intracellular ROS in HP-treated tissues were detected following 2-hour postincubation: 2.4- and 3.0-fold increases for tissues treated with 20 mM and 50 mM, respectively. Interestingly, the barrier recovered in tissues treated with 20 mM HP after 24 hour post-incubation while 17

tissues treated with 50 mM HP deteriorated in both barrier permeability and tissue morphology following 24 hour post-incubation (Fig. 7A, G1 and G2). Dose dependent disruption of apical layers was observed in H&E-stained histological cross-sections (Fig. 7G) which corresponds to the decreased barrier. Also, HP-treated tissues displayed reduced viability as measured by mitochondrial activity (MTT assay, Fig. 7B) and increased cytotoxicity as measured by membrane integrity (LDH release, Fig. 7C). Similarly, elevated level of lipid peroxidation, as a detected by 8-ISO release, was observed for both conditions with and without 24 hour post-incubation (Fig. 7E). Significantly increased secretion of the pro-inflammatory cytokine, IL-8, was detected for both treatments, but only following 24 hour postincubation (Fig. 7F). The most pronounced effects in response to NM treatment were lipid peroxidation and IL-8 release following 24 hours post-exposure. 10 min and 30 min treatments with 7.8 mM of NM followed by a 2 hour post-incubation lowered tissue barrier to 71.5±0.7% and 78.9±9.3%, respectively, but didn’t affect tissue viability (Fig. 7A-C), as confirmed by H&E stained tissue morphology (Fig. 7G1). Notably, tissues that were exposed to NM for a short time (10 min) and then post-incubated for 24 hours recovered its barrier property as confirmed by tissue histology. On the other hand, tissues that were exposed to NM for a longer time (30 min) had remarkably weaker barrier (41.0±3.3%), increased cytotoxicity, and poor morphology (Fig. 7A-C and G2) following 24 hour post-incubation. Interestingly, both conditions had a powerful effect on lipid peroxidation and IL-8 release following 24 hour postincubation; however, 10 min exposure led to stronger increases of lipid peroxidation (20.1-fold) and IL-8 release (18.6-fold) when compared to untreated control tissues (Fig. 7E and F). 2.5. Oxidative Stress-Induced Gene Expression Profile in the Reconstructed 3D-HCE Tissues One of the goals of this work was to find what genes are regulated in corneal epithelium in response to oxidative stress. Utilizing a PCR gene array, we analyzed the expression of 84 OSresponsive genes and found specific groups of genes that are regulated in response to different mechanisms of OS-induced corneal injury (Fig. 8 and Table 2). Specifically, we analyzed gene expression in 3D-HCE tissues after exposure to severe DED conditions (DSC3, 38°C, 60% RH) for 24 18

hours; exposure to full spectrum solar irradiation equivalent to 120 mJ/cm2 of UVB; topical exposure to 50 mM of HP for 2 hours; and topical exposure to 7.8 mM of NM for 30 min. All the treatments were chosen to induce physiologically relevant injuries and to avoid extensive tissue damage by necrosis. Gene expression analysis of 3D tissues was performed following 24 hours post-incubation to allow for the development of the damaging effect. Of 84 genes related to the OS responses that were analyzed (including peroxidases, superoxide disumutases, antioxidants, oxygen transporters, and genes involved in ROS metabolism), 8 genes were below the detection limit in the negative control 3D-HCE tissues. All treatments resulted in modest alterations in gene expression that were specific to the mechanism of the OS damage (Fig. 8). It was shown that Taq-Man real time PCR array has acceptable sensitivity and accuracy in detecting differential expression for genes with more than 2-fold changes (Wang et al., 2006). Therefore, a 2-fold change was used as a cutoff to determine differential gene expression in OS-generated corneal injuries. Of the 84 genes studied, only one gene, SPINK1, serine peptidase inhibitor, a known OS pathway activity signature gene, was up-regulated in all OS-generated injuries and HMOX1, oxygenase, was up-regulated in all conditions except in the NM-treated tissues (Table 2). Incubation of 3D-HCE tissues under severe DED condition (DSC3) for 24 hours induced >2-fold gene upregulation of peroxidases (GPX5 and GPX2), cyclooxygenase (PTGS2), superoxide metabolism responsive gene lypoxygenease (ALOX12), and the OS pathway activity signature genes BAG2 and NOS2 (Table 2). The DED model resulted in down-regulation of more genes than any other condition, including antioxidant GSTZ1, peroxidase EPX, oxygen transporter MB, genes involved in ROS metabolism (surfactant protein SFTPD, chemokine CCL5, hydrolase EPHX2, keratin KRT1), as well as the OS pathway activity signature genes, uncoupling protein UCP2, phosphatase LHPP, and trafficking protein particle TRAPPC6A. UVB irradiation (120 mJ/cm2) upregulated SPINK1, HMOX1, cytoglobin CYGB, reductase TSNRD1, peroxidases GPX5, GPX2, and GPX3, NCF1 oxidase, and metallothionein MT3 (Table 2). Reductase MSRA, chemokine CCL5, and selenoprotein P (SEPP1) were > 2-fold down-regulated following exposure to solar radiation. Oxidative stress induced by HP treatment (50 mM) for 2 hours led to the fewest changes in the gene expression that could be detected 19

following 24-hour post-treatment (Fig. 8C and Table 2). Of the panel OS-responsive genes studied, only serine peptidase inhibitor SPINK1, oxygenase HMOX1, and antioxidant peroxidase GPX3 were found to be >2-fold up-regulated in response to HP treatment followed by 24 hour post-incubation. Exposure to NM also led to activation of SPINK1, but oxygenase HMOX1 was unchanged. exposure and 24 hour incubation resulted in

>2-fold up-regulated

NM

of cyclooxygenase (PTGS2),

selenoprotein P (SEPPP1), and sulfiredoxin (SRXN1), ligase GCLM, heat shock protein HSPA1A, and oxidase NCF1 and >2-fold down-regulation of antioxidants cyclooxygense (PTGS1), apolipoprotein (APOE), catalse (CAT), and reductase (GSR), as well as the nuclear receptor co-activator (NVOA7) genes involved in ROS metabolism transcription factor FOXM1, cytoskeletal reorganization PDLIM1, and keratin 1 KRT1. 3. Discussion The availability of an in vitro 3D human corneal epithelial tissue model that is similar to native corneal epithelium is an important step towards facilitating success in the ophthalmic drug discovery. This manuscript describes mature in vitro reconstructed 3D-HCE tissues that structurally resemble human corneal epithelium (Fig. 1C and D) and express site-specific cytokeratins and mucins (Fig. 2 and Table 1). Adequate mucin expression is an essential factor for the protective function of the tissue barrier (Table 1) (Ablamowicz and Nichols, 2016). The 3D-HCE tissues contain tight junctions (Fig. 3) and desmosomes (Kaluzhny et al., 2018), and develop barrier properties with TEER above 900 Ω·cm² (Fig. 4), similar to the values obtained using in vivo intraocular electrodes (Uematsu et al., 2016). There is only limited information available about in vivo TEER in normal human corneas, due to the nature of the procedures necessary to assess corneal epithelial barrier. Using a novel non-invasive technique, TEER values of 750 ± 111 Ω·cm² and 690 ± 69 Ω·cm² for rabbits and humans, respectively, have been reported (Uematsu et al., 2015; Uematsu et al., 2016). Other reports presented somewhat higher TEER values (1122.3 ± 61.3 Ω·cm²) for excised rabbit cornea (Nakamura et al., 2010). TEER is a useful technique for detecting changes in the barrier function without interrupting an experiment. Alteration of the tissue barrier is a very sensitive measure and indicative of changes to superficial cells and tight junctions within the tissue. 20

Oxidative stress and the associated corneal damage have been linked to a variety of inflammatory conditions, including ocular surface injuries and dry eye disease (Buddi et al., 2002; Cejkova and Cejka, 2015; Yamaguchi, 2018). In this study, we investigated the utility of the in vitro reconstructed 3D corneal epithelial tissue model to discriminate between different mechanisms of corneal injuries induced by exposure to various oxidative stress inducing conditions. Reconstructed 3D corneal epithelial tissues growing inside cell culture inserts are easily handled with a use of sterile forceps and experiments are performed in standard 6-well or 12-well plates. The use of cell culture inserts allows both topical and basolateral tissue treatments (Fig. 1A and B). Due to its location and function, the cornea is exposed to sunlight and atmospheric oxygen which produce ROS making corneal epithelial cells susceptible to oxidative stress (OS). To withstand OS, the cornea has developed several detoxification mechanisms, including expression of key enzymes, such as aldehyde dehydrogenases (ALDH1A1 and ALDH3A1) and thioredoxin reductase (TXNRD1) that metabolize aldehydes. In addition, 4-Hydroxynonenal (4-HNE) plays a role in selenium metabolism to withstand the chain of reactions induced by OS, ROS, and lipid peroxidation (Allen and Tresini, 2000; Buddi et al., 2002; Han et al., 2008; Wojcik et al., 2013). In fact, ALDH3A1 expression is remarkably high in the cornea of mammalian species, comprising from 5 to 50% of soluble protein content, but is almost absent from the cornea of other vertebrates (Cejkova and Cejka, 2015; Estey et al., 2007).

Therefore, in vivo-like expression of tissue-specific corneal detoxification enzymes is

essential to adequately model the in vivo response. As shown in Table 1, gene expression for key corneal detoxification enzymes for the 3D-HCE tissues is comparable to that of excised corneal epithelial tissue, indicating that in vitro reconstructed 3D-HCE tissues are well equipped for proper modeling of responses to oxidative stress. There is a growing body of evidence that chronic exposure to oxidative stress is involved in the initiation and progression of cellular injury that may lead to ocular surface pathology in DED (Cejkova and Cejka, 2015; Pinazo-Duran et al., 2014). We exposed the in vitro reconstructed 3D-HCE tissues to desiccating stress conditions (DSC) of various severities: increased temperature, decreased humidity, or both to generate a physiologically relevant epithelial DED model without inducing non-physiologic 21

damage of the tissues. We have demonstrated that increased severity of DSC leads to more rapid and more severe effects on the epithelial tissue function (Fig. 5). All DSC settings led to gradual epithelial tissue dehydration and cornification that correlated with in vivo changes observed in DED (Cejkova and Cejka, 2015; Chen et al., 2008; Meloni et al., 2011). Incubation of 3D-HCE tissues at severe DSC settings (DSC3, 38°C, 60%RH) led to more rapid tissue dehydration (Fig. 6F) that coincided with reduced TEER and tissue viability (Fig. 5). Morphologically, 3D-HCE tissue incubated at DSC3 for 72 hours did not resemble ocular epithelial tissue, therefore results were not included in the study. It has been reported that morphological changes of DED coincide with the accumulation of ROS and chronic inflammation; and the release of cytokines and chemokines initiate and amplify the inflammatory responses (Buddi et al., 2002; Cejkova and Cejka, 2015; Pinazo-Duran et al., 2014). IL-8, a chemokine normally detected at the site of inflammation, was released not only after exposure to DSC, but also after UVB irradiation, hydrogen peroxide and nitrogen mustard exposure (Fig. 7).

We

used 8-Isoprostane (8-ISO), a biomarker for oxidative stress, to indirectly measure accumulation of ROS. 8-ISO has been shown to be produced independently of cyclooxygenase enzymes, be a stable lipid peroxidation product and a useful biomarker of the OS (Fig. 5D, 6D, and 7E) (Cejkova and Cejka, 2015; Milne et al., 2015; Samar, 2007; Zheng et al., 2013). Artificial tears do not have pharmacological activity but they increase hydration and improve lubrication of the ocular surface. As expected, topical administration of over-the-counter lubricating eye drops (GenTeal, Alcon) considerably protected the 3D tissues from severe DSC3 and decreased the morphological and molecular changes. Improved 3DHCE tissue hydration led to improved tissue morphology, barrier properties, and mucin expression (Fig. 6G). Topical application of the eye drops also reduced ROS accumulation as shown by decreased 8ISO accumulation (Fig. 6D) and lower release of the pro-inflammatory cytokine, Interleukin-8 (IL-8, Fig. 6E). Responses of the 3D-HCE tissues to UV irradiation, hydrogen peroxide, and nitrogen-mustard induced corneal epithelial injury resembled that of DED tissues, except each had injury-specific morphological and molecular outcomes (Fig. 7). UV irradiation led to intracellular ROS accumulation and 8-ISO release into the medium but it did not have an effect on tissue structure, barrier and viability. 22

Exposure to 50 mM of hydrogen peroxide solution led to corneal barrier impairment, significant IL-8 release, decreased tissue viability and morphological changes following 24 hours post-incubation, whereas 20mM-treated tissues appeared normal and recovered following the post-incubation. While both 10 and 30 min exposures to 7.8 mM of nitrogen mustard solution led to barrier impairment, increased cytotoxicity, vast lipid peroxidation and IL-8 release; altered tissue morphology was apparent only for longer exposure time. In healthy corneas, the antioxidant enzymes involved in the reduction-oxidation homeostasis lessen the negatives effects of the oxidative stress. Cells are typically able to overcome mild oxidative stress and restore homeostasis, but severe oxidative stress may trigger apoptosis, necrosis, and cell death (Cejkova and Cejka, 2015; Deng et al., 2015). When threshold levels of UV radiation or other oxidative exposures are exceeded, corneal instability appears. In this study, the exposure of 3D-HCE tissues to full spectrum radiation corresponding to 60 mJ/cm2 and 120 mJ/cm2 induced oxidative stress as confirmed by ROS and 8-ISO accumulation. However, the innate reduction-oxidation system was able to counteract the OS as confirmed by unchanged tissue viability, morphology, and barrier function (Fig. 7). Likewise, the exposure of 3D-HCE tissues to the oxidizing agents HP and NM led to OS, as shown by accumulation of 8-ISO and release of IL-8 in a time-dependent manner. The 3D-HCE tissues that were exposed to low doses of the insult (20 mM of HP and 10 min exposure to NM) were able to recover and to maintain normal tissue morphology, viability, and the barrier property. However, the tissues that were exposed to high doses of the insult (50 mM of HP and 30 min exposure to NM) could not overcome the insult and epithelial tissue morphology, tissue viability, and barrier property progressively worsened. Our data are in agreement with a study that reported cellular injury and oxidative damage induced in immortalized HCE cells in monolayer culture and rat corneas by exposure to 0.25 mM of HP (Yin et al., 2018). Time-dependent decreases in cell viability, upregulated production of ROS and enzymes responsible for ROS generation (NOX4), and downregulation of factors responsible for ROS degradation (superoxide dismutase, catalase, and glutathione S-transferase P) were observed. One

23

important drawback of the HCE study was that the HCE cultured cells could not be treated with physiologically relevant concentrations and/or exposures similar to the animal model. Clinically, eye injury due to nitrogen mustard (a potent alkylating agent) exposure, displayed a delayed biphasic response that initially included photophobia and inflammation, followed by corneal ulceration, chronic inflammation, and often the loss of vision (Goswami et al., 2016a; Zheng et al., 2013). NM studies in ex vivo rabbit corneal organ cultures demonstrated increased epithelial thickness, epithelial detachment, apoptotic cell death and caused increased expression of cyclooxygenase-2 (COX-2) (similar to our observation in 3D-HCE tissues, Fig. 7G and Table 2), a key player involved in the prostaglandin synthesis and inflammatory responses (Goswami et al., 2016a; Goswami et al., 2016b; Zheng et al., 2013). It was previously shown that the injured or diseased corneas have decreased antioxidant capacity, while the levels of pro-oxidants remained at physiological levels or even increased, leading to the antioxidant/pro-oxidant imbalance and oxidative stress (Cejkova and Cejka, 2015). Under oxidative stress, cells try to counteract the effects by decreasing or increasing the expression of specific genes (e.g. defensive enzymes, transcription factors, and structural proteins), leading to tissue- and injuryspecific changes on the molecular level (Allen and Tresini, 2000). Utilizing a PCR gene array, we compared the effects of OS-induced corneal epithelial damage on the expression of 84 OS-responsive genes and found specific molecular footprints for each mechanism of OS-induced injury (Fig. 8). Significant changes in the expression of 32 genes were observed. It was previously shown that the expression of OS-related genes depended on the severity of the exposure and the time elapsed between the exposure and the gene analysis (Han et al., 2008). It is also possible that the expression of some genes affected by OS was normalized following 24 hours of post-incubation. In this study, all conditions resulted in significant activation of SPINK1 gene expression but the reason for this is unknown (Table 2). SPINK proteins are expressed in diverse tissues and act through regulation of serine proteases to prevent imbalances in protease activities (Mehner and Radisky, 2019; Weber et al., 2017). It has been previously demonstrated that the balance between proteases and 24

protease-inhibitors system plays a key role in maintaining cellular and tissue homeostasis and its dysregulation may lead to a number of ocular diseases (Pescosolido et al., 2014). A comparison of the amino acid sequences of SPINK1 and epidermal growth factor (EGF) revealed significant similarity, and based on this finding, it has been proposed that SPINK1 may function as an autocrine or paracrine growth factor (Mehner and Radisky, 2019). Mice injected with diquat to induce OS led to upregulation of the antioxidant gene for the potent ROS scavenger, metallothionein 1 (MT1). Significant increases in thioredoxin reductase 1 (TXNRD1), selenoproteins, and surfiredoxins were also observed (Han et al., 2008). In the current study, UVBinduced OS led to significant upregulation of MT3 and TXNRD; however, the other conditions didn’t induce changes in metallothionein or TXNRD1 gene expression (Table 2). In addition, selenoprotein SEPP1 and sulfiredoxin SRXN1 were upregulated in 3D-HCE tissues treated with NM for 30 min (Table 2). PTGS2, also known as inducible cyclooxygenase (COX-2), is the key enzyme in prostaglandin biosynthesis, and is responsible for the prostanoid biosynthesis involved in inflammation and mitogenesis. In our study, PTGS2 gene upregulation was detected in the 3D-HCE tissues cultured under DSC conditions for 24 hours and following 30 min treatment with NM and 24-hour post incubation. In this study, we have shown that four types of glutathione peroxidases (GPX) are highly expressed in the human corneal epithelium and in vitro reconstructed 3D-HCE tissues (Table 1). GPX participate in the reduction of hydrogen and lipid peroxides and play a major physiological role in the regulation of oxidative stress. All conditions except NM-treatment induced increased levels of at least one type of peroxidase gene (Table 2). Mammalian GPX1 to GPX4 have been shown to be seleniumcontaining enzymes and require a steady supply of selenium to maintain the enzyme activity. Selenoprotein P (SEPP1), also known as a selenium-transfer plasma glycoprotein, was shown to be secreted in tear fluid and its concentration was reduced in dry eye patients (Higuchi et al., 2012). In 3DHCE tissues SEPP1 was overexpressed in NM-treated tissues and downregulated following UV irradiation (Table 2).

25

Cytoglobin (CYGB), a ubiquitously expressed hemoglobin that may facilitate respiration and the storage and transport of oxygen, was also shown to scavenge nitric oxide or reactive oxygen species, or serve a protective function during oxidative stress (Reeder et al., 2011). CYGB was overexpressed in the UV-exposed 3D tissues (Table 2). Neutrophil cytosol factor 1 (NCF1) is a 47 kDa cytosolic subunit of neutrophil NADPH oxidase (NOX).

This oxidase is a multicomponent enzyme that is

activated to produce superoxide anion. Genetic variability in the NCF1 gene has been found to be associated with Sjögren's syndrome (Zhao et al., 2017). NCF1 was overexpressed in the UV-exposed and NM-treated 3D-HCE tissues (Table 2). 4. Conclusion In conclusion, we have successfully developed an in vitro reconstructed 3D-HCE tissue model that is comparable to the in vivo human corneal epithelium when exposed to various oxidative stress conditions.

This is an important development because changes to the ocular surface are difficult to

study in vivo. Utilization of the reconstructed 3D corneal epithelial tissue model allows in vivo-like exposures, administration of physiologically relevant concentrations, and analysis immediately following administration of a material.

Other advantages of the in vitro 3D-HCE tissue model include the

possibility of high throughput screening of drug candidates and access to both the apical and basolateral sides. The in vitro reconstructed 3D-HCE tissues will make pharmacological studies rapid, will circumvent excessive use of animals, and allow for basic studies related to corneal epithelial physiology, oxidative stress, and ocular injury. Studies using the in vitro corneal epithelial tissue model may suggest biomarkers for oxidative stress and vesicating agents and help identify new compounds and antioxidants to treat corneal injuries from vesicants or other OS-inducing chemical agents. We anticipate that the reconstructed 3D corneal epithelial tissue model will facilitate the research and development of ocular therapeutics to address oxidative and chemical insult to the cornea.

26

Table 1. Gene expression of surface mucins and key detoxification enzymes

in corneal epithelium and 3D human corneal epithelial tissue model (3D-HCE) Symbol MUC1 MUC4 MUC16 ALDH1A1 ALDH3A1 GPX1 GPX2 GPX3 GPX4 MGST1 MGST2 MGST3 GSTT1 GSTA4 GSTM2 GSTM3 TXNRD1

Description Cell surface associated mucin Aldehyde dehydrogenase

Glutathione peroxidase

Microsomal glutathione S-transferase

Glutathione S-transferase

Corneal epithelium Gene expression 2 comment 0.0086 low 0.0002 very low 0.0256 moderate 1.1494 very strong 3.3886 very strong 1.6101 very strong 0.3383 strong 0.0022 low 1.8001 very strong 0.1757 strong 0.4865 strong 0.7548 strong 0.1273 strong 0.5399 strong 0.0447 moderate 0.0049 low 0.0417 moderate -∆Ct

Thioredoxin reductase UDPUGT1A1 0.0004 very low glucuronosyltransferase MT2A Metallothionein 1.2057 very strong PKM Pyruvate kinase 3.4814 very strong Catechol-OCOMT 0.6633 strong methyltransferase -∆Ct -∆Ct Gene expression comment: 1.0 ≤ 2 : very strong expression; 0.1 ≤ 2 < 1.0: -∆Ct -∆Ct < 0.01: low expression; 0.0001 ≤ 2 < 0.1: moderate expression; 0.001≤ 2 standard deviation.

0.0381 0.0002 0.0479 1.4048 0.1990 1.9997 0.0565 0.0089 0.6964 1.2836 0.4923 0.2472 0.0773 0.4390 0.0232 0.0806 0.0161

3D-HCE Gene expression comment moderate very low moderate very strong strong very strong moderate low strong very strong strong strong moderate strong moderate moderate moderate

0.0019

low

1.7241 3.6015

very strong very strong

0.6970

strong

2

-∆Ct

-∆Ct

strong expression; 0.01 ≤ 2 < 0.001: very low expression; SD,

27

Table 2. Regulation of oxidative stress related genes in the reconstructed 3D human corneal epithelial (3D-HCE) tissues in response to DED conditions, UV irradiation, exposure to Hydrogen peroxide, and Nitrogen-mustard. Genes with more than 2-fold up- or downregulation are presented. Gene symbol GSTZ1 PTGS1 (COX1) PTGS2 (COX2)

Description

DED

UVB

H2O2

NM

Function

Glutathione transferase Antioxidant

Cyclooxygenase

MB

Myoglobin

CYGB

Cytoglobin

Oxygen Transporters Oxygen Transporters; ROS metabolism

BAG2

BCL2-associated

NOS2

iNitric oxide synthase

LHPP

Phosphatase

NOX5

NADPH oxidase

OS Pathway Activity Signature Gene

TRAPPC6A

Trafficking protein particle

NCOA7

Nuclear receptor co-activator

SPINK1

Serine peptidase inhibitor

HMOX1

Oxygenase

GCLM

Ligase

TXNRD1

Reductase

UCP2

Uncoupling protein

SFTPD

Surfactant protein

MSRA

Reductase

OS Pathway Activity Signature Gene; ROS metabolism OS Pathway Activity Signature Gene; ROS metabolism; Antioxidant OS Pathway Activity Signature Gene; Superoxide metabolism

CCL5

Chemokine

EPHX2

Hydrolase

HSPA1A

Heat shock protein

FOXM1

Transcription factor

PDLIM1

Cytoskeletal reorganization

KRT1

Keratin

SEPP1

Selenoprotein P

ROS metabolism

GPX5 GPX2 EPX

Peroxidase ROS metabolism; Antioxidant

GPX3 SRXN1

Sulfiredoxin

APOE

Apolipoprotein

CAT

Catalase

GSR

Reductase

ALOX12

Lipoxygenase

NCF1

Oxidase

MT3

Metallothionein

Superoxide metabolism Superoxide metabolism; Antioxidant

updownno change regulated regulated Keys: OS, oxidative stress; ROS, reactive oxygen species; DSC, desiccating stress conditions; H2O2, hydrogen peroxide; NM, nitrogen-mustard Color keys:

28

Figure 1. Diagram and morphology of the 3D human corneal epithelial (3D-HCE) tissue model. A. Diagram of the 3D-HCE tissue model grown on microporous membrane cell culture inserts at the air liquid interface (ALI). This system allows both topical exposure (onto the tissue surface) and systemic exposure (into the medium) of test materials. B. Handling of the inserts containing the 3D-HCE tissues under ALI culture conditions. C. Hematoxylin and eosin stained cross-section of 3D-HCE tissues. D. Hematoxylin and eosin stained cross-section of human corneal tissue (corneal button).

B

A

C

D

29

Figure 2. Immunohistochemical analysis for corneal markers in the 3D-HCE tissue model. Cross sections of 3D-HCE tissues stained for: Cytokeratins CK3/12 (red) (A and B) and Aldehyde Dehydrogenase-3 (ALDH3A1, green) (C and D). 400X magnification. Counter stain nuclear stain DAPI (blue). Dashed red line indicates the position of the microporous membrane.

A

C

B

D

30

Figure 3. Immunohistochemical analysis for tight junction proteins in the 3D-HCE tissue model. Topical view of 3D-HCE tissues, confocal microscopy: ZO-1 (green) (A and B), occludin (red) (C and D), and claudin-1 (green) (E and F). A and C - F. 200X magnification; B. 600X magnification; Counter stain nuclear stain DAPI (blue) (B, D, and F)

A

B

C

D

E

F

31

Figure 4. Transepithelial Electrical Resistance (TEER) of 3D-HCE tissues model during development (up to day 10), before shipping (day 10), after simulated shipping and 1 hour equilibration (day 11), and extended incubation (days 12–15). Average TEER value with standard deviation from a minimum of n=3 tissue lots is presented. Dashed line indicates TEER of the mature 3D-HCE cultures prior to the shipping and following the simulated shipping and pre-equilibration.

1600 1400

TEER (Ω·cm2)

1200 1000 800 600 400 200 0

5

6

7

8

9

10

11

12

13

14

15

Days in culture

32

Figure 5. In Vitro Dry Eye Disease (DED) Corneal Epithelial Model. The reconstructed 3D-HCE tissues were placed under a range of desiccated stress conditions (DSC): mild - DSC1: 37°C/80%RH, moderate - DSC2: 37°C/60%RH, and severe - DSC3: 38°C/60%RH for up to 72h. Effect on barrier property (TEER) (A); tissue viability (MTT assay) (B) cytotoxicity (LDH release) (C); 8-Isoprostane (D) and IL-8 (E) released in the medium by 3D-HCE tissues. * p≤0.05; **p≤0.001. SCC, standard culture conditions; DSC, desiccating stress conditions. 120 24h

120 48h

72h

Barrier (TEER, % to NC)

100

* *

**

80

**

60

** 40 20

100

Viabilty (MTT, % to NC)

24h

SCC 37°C 95%RH

DSC1 37°C 80%RH

DSC2 37°C 60%RH

DSC3 38°C 60%RH

60 40 20

SCC 37°C 95%RH

DSC1 37°C 80%RH

DSC2 37°C 60%RH

DSC3 38°C 60%RH

2500 24h

48h

72h

*

100

24h

*

48h

**

72h

2000

*

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Figure 6. Severe In Vitro Dry Eye Disease (DED) Corneal Epithelial Model. The reconstructed 3DHCE tissues were placed under severe DED conditions (DSC3, 38°C, 60% RH, and 5% CO2) for up to 48h. 25 µl of commercial eye drops (GenTeal, Alcon) were applied topically every 24 hours. A. Barrier properties (TEER); B. Tissue viability (MTT assay); C. LDH Release; D. Accumulation of 8-Isoprostane in the medium after 24 hours; E. IL-8 released into the medium after 24 hours; F. Tissue histology (H&E staining of cross-sections). G. Topical view of 3D-HCE tissues; MUC1- staining (red), 10x objective. SCC, Standard Culture Conditions (Control); DSC, Desiccating Stress Conditions; * p≤0.05; **p≤0.001 120

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Figure 7. Response of 3D-HCE tissues to harmful environmental conditions. Full spectrum solar light simulator delivered UV irradiation at doses equivalent to 60 mJ/cm2 and 120 mJ/cm2 of UVB. Hydrogen peroxide (HP), and nitrogen-mustard (NM) were applied topically on the surface of the 3DHCE tissues. A. Effect on barrier property (TEER), B. Tissue viability (MTT assay) and C. Cytotoxicity (LDH release). D. Accumulation of intracellular ROS after 2h post-incubation. Release into medium of E. 8-Isoprostane and. F. IL-8. G. 3D-HCE tissue histology (H&E staining of cross-sections) after 2h (G1) and after 24h (G2) post-incubation. * p≤0.05; **p≤0.001

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Figure 8. Evaluation of the oxidative stress-related gene expression in the reconstructed 3DHCE tissues. The scatter plots compare gene expression levels, log10 (2-∆Ct), between treated and non-treated 3D-HCE tissues. (A) Incubation at severe DED conditions (DSC3, 38°C, 60% RH) for 24h; (B) Exposure to the full spectrum solar irradiation (120 mJ/cm2 of UVB) and 24h post-incubation; Topical application of: (C) Hydrogen peroxide solution (50 mM for 2h) and 24h post-incubation or (D) nitrogen-mustard solution (7.8 mM for 30 min) and 24h post-incubation. Log 10 (UVB, 2-∆∆ Ct)

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Oxidative Stress in Corneal Injuries of Different Origin: Utilization of 3D Human Corneal Epithelial Tissue Model. Corresponding author: Yulia Kaluzhny, [email protected], 1-508-881-6771, x229 Highlights •

Physiologically relevant organotypic normal human corneal epithelial tissue model

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Realistic exposures to environmental factors: low humidity, UV, harmful agents

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3D tissues reproduce key features of oxidative stress–induced ocular damage

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Specific cellular and molecular responses to different types of oxidative stress

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Regulation of oxidative stress-responsive genes following an oxidative damage