Ectoine Enhances Mucin Production Via Restoring IL-13/IFN-γ Balance in a Murine Dry Eye Model

Abstract

Purpose: This study aimed to explore protective effects and potential mechanism of ectoine, a natural osmoprotectant, on ocular surface mucin production in dry eye disease.

Methods: A dry eye model was established in C57BL/6 mice exposed to desiccating stress (DS) with untreated (UT) mice as controls. DS mice were topically treated with 2.0% ectoine or PBS vehicle. Corneal epithelial defects were assessed by Oregon Green Dextran (OGD) fluorescent staining. Conjunctival goblet cells, ocular mucins, and T help (Th) cytokines were evaluated by immunofluorescent staining or ELISA, and RT-qPCR.

Results: Compared with UT mice, corneal epithelial defects were detected as strong punctate OGD fluorescent staining in DS mice with vehicle, whereas ectoine treatment largely reduced OGD staining to near-normal levels. Conjunctival goblet cell density and cell size decreased markedly in DS mice, but was significantly recovered by ectoine treatment. The protein production and mRNA expression of two gel-forming secreted MUC5AC and MUC2, and 4 transmembrane mucins, MUC1, MUC4, MUC16, and MUC15, largely decreased in DS mice, but was restored by ectoine. Furthermore, Th2 cytokine IL-13 was inhibited, whereas Th1 cytokine IFN-γ was stimulated at protein and mRNA levels in conjunctiva and draining cervical lymph nodes (CLNs) of DS mice, leading to decreased IL-13/IFN-γ ratio. Interestingly, 2.0% ectoine reversed their alternations and restored IL-13/IFN-γ balance.

Conclusions: Our findings demonstrate that topical ectoine significantly reduces corneal damage, and enhances goblet cell density and mucin production through restoring imbalanced IL-13/IFN-γ signaling in murine dry eye model. This suggests therapeutic potential of natural osmoprotectant ectoine for dry eye disease.

All mucosal tissues expose a wet epithelial surface covered by a mucous coating known as the glycocalyx formed with highly diverse glycoproteins and glycolipids. Mucins, a family of large high molecular weight (0.2 to 40 million Dalton [Da]) glycoproteins, are the main structural components of mucus. Mucins cover the surfaces of the respiratory, digestive, gastrointestinal, and genitourinary tracts, protecting epithelial cells from infection, dehydration, and physical or chemical damage, providing protection and lubrication for the epithelial surface. The expression of each mucin is organ- and cell-specific. Alterations in mucin expression, glycosylation, or localization have been seen in a variety of pathological conditions, such as cancers, inflammatory bowel disease, and ocular disease (see review articles^1–3). 

A total of 21 mucin genes (MUC) have been identified in humans, and their products can be classified into 2 types: membrane-associated or transmembrane (MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13, MUC14, MUC15, MUC16, MUC17, MUC20, MUC21, and MUC22), and secreted mucins based on their structural difference and distribution. Secreted mucins are further subclassified to large gel-forming (MUC2, MUC5AC, MUC5B, MUC6, and MUC19) and small soluble mucins (MUC7, MUC8, and MUC9) types. Collectively, these mucins contribute to regulating the host-environment interactions at mucosal surfaces through various mechanisms ranging from forming physiochemical barriers to regulating signal transduction pathways in epithelial.^1–4 

Ocular surface mucins are synthesized by corneal and conjunctival epithelia and lacrimal gland.^4–7 These mucins play a vital role in maintaining ocular surface homeostasis by lubricating and protecting mucosal epithelial cells from abrasive stress, desiccation, and potential pathogens. Mucins are present in the tears where they maintain hydration. Mucins also provide immune-modulatory functions for strengthening the epithelial barrier, acting as a scaffold for many antimicrobial factors, preventing binding of pathogens to the ocular surface, and clearing away pathogens and other debris.^6,8,9 

Secreted mucins, including gel-forming and soluble, are major and are produced by specialized goblet cells. Secreted mucins create a viscous mucous layer over the ocular surface epithelium. In contrast, the aqueous and mucin components of the tear film combine to create a single muco-aqueous gel, serving to reduce evaporation. 

Transmembrane mucins are the distinguishing components of the mucosal glycocalyx. At the ocular surface, they maintain wetness, lubricate the blink, stabilize the tear film, and create a physical barrier to the outside world. In addition, it is increasingly appreciated that transmembrane mucins function as cell surface receptors that sense the extracellular environment and transduce signals intracellularly. In various mucosal tissues, transmembrane mucins activate or inhibit intracellular signaling cascades that regulate inflammation, cell-cell interactions, differentiation, and cell death.^4,6,7,10 

Mucin abnormalities have been observed in various ocular surface disease, such as dry eye,^8,11,12 contact lens wear and infection,^13,14 allergy,^15,16 pollution, pterygium, and ocular rosacea.^8,9 Dry eye is a common ocular inflammatory disease with increasing prevalence worldwide,^17,18 Patients with dry eye often suffer ocular discomfort, irritation, and visual disturbance, which reduce the quality of life of the patient.¹⁹ Loss of goblet cells, consistent decreases in MUC5AC and altered expression of transmembrane mucins, including MUC1, MUC4, and MUC16, with unstable tear film have been observed in both patients with Sjögren syndrome and non-Sjögren dry eye,^8,20,21 as well as in murine dry eye models.^22–24 Mucins are recognized as therapeutic targets for dry eye disease.^23,25,26 However, ocular mucins remain relatively under-studied and inadequately characterized. Development of a new line of treatment targeting the mucin shortage is important for improving the treatment of dry eye disease. 

Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid), a compatible water molecule-binding solute, is a natural osmoprotectant produced by certain bacterial species living in extreme conditions.²⁷ Ectoine has a unique effect of strong preferential hydration through its reorganization of water molecules.²⁸ Ectoine was found to inhibit the inflammatory responses in chronic inflammatory diseases.²⁹ For ocular disease, ectoine has been used in clinical trials to treat dry eye disease^30,31 and seasonal allergic rhinoconjunctivitis.^31,32 However, these clinical trials of ectoine topical application are limited in a few European countries, such as Russia, Ukraine, Switzerland, and Germany, but not in the United States; and the investigations were restricted to clinical efficacy on dry eye symptoms and signs. There is almost no animal study on ectoine. The multifunctional therapeutic effects and molecular mechanism of ectoine in dry eye are largely unknown. The potential role and mechanism of ectoine in protecting goblet cells and mucin production associated with dry eye disease remain poorly understood. 

Our team recently investigated the novel role and molecular mechanism of ectoine in protecting corneal epithelial cell viability and barrier integrity in primary human corneal epithelial cells exposed to hyperosmotic stress, an in vitro dry eye model.³³ We also observed that ectoine prevents corneal epithelial damage and reduces ocular inflammation in a murine model of experimental dry eye induced by desiccating stress.³⁴ In the present study, we further explored the novel effects and potential mechanism of ectoine on ocular surface mucin production in a dry eye mouse model. 

Materials and Methods

Animals

The animal research protocol was approved by the Institutional Animal Care and Use Committee, Center for Comparative Medicine, Baylor College of Medicine, and it conformed to the standards in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals,³⁵ and ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

C57BL/6J mice were purchased from the Jackson Laboratories (Bar Harbor, ME, USA) and housed in specific pathogen-free conditions in microisolator cages in the facilities of Baylor College of Medicine. Mice were kept on diurnal cycles of 12 hours/light and 12 hours/dark with ad libitum access to water, food, and environmental enrichment. 

Murine Model of Experimental Dry Eye Disease

A murine model of experimental dry eye disease was induced by desiccating stress (DS) in female C57BL/6 mice using our previously published methods.^36,37 In brief, mice aged 8 to 10 weeks were exposed to an air draft and low ambient humidity (20–30%) in the environment controlled Darwin Chambers with oral scopolamine hydrobromide (Sigma-Aldrich, St. Louis, MO) in drinking water (0.5 mg/mL) to inhibit tear secretion for 5 consecutive days. Control mice were maintained in a normal environment at 50% to 75% relative humidity. 

Ectoine Topical Treatment in DS Mice

The dry eye model mice (DS) were topically treated by instillation of 5 uL/eye of 2% ectoine (provided by AbbVie, Inc., North Chicago, IL, USA) in phosphate buffered saline (PBS), or PBS vehicle, 4 times daily for 5 days during exposure to desiccation.³⁴ For normal controls, the age- and gender-matched untreated (UT) mice were maintained at 50% to 75% humidity in a normal environment. On day 5, clinical signs of dry eye and corneal damage were assessed by a stereo microscopy and Oregon Green dextran (OGD) fluorescein staining. To evaluate the expression and production of ocular goblet cells, mucins, and cytokines, whole eyeballs, corneal epithelium, conjunctival tissues, and ocular surface draining cervical lymph nodes (CLNs) were collected after animal euthanasia. 

In each group of each experiment, two eyeballs from different mice were embedded in optimal cutting temperature (OCT) compound for cryosections to be used for immunofluorescent staining; another two eyeballs from different mice were used for paraffin embedding for PAS staining; corneal epithelium from four eyes, and conjunctival tissues from two eyes were collected into RNA lysis buffer in one tube as one sample for total RNA isolation and RT-qPCR; conjunctiva from another two eyes were collected in protein lysis buffer as one sample for ELISA; and CLNs were collected from three mice were embedded in OCT to prepare cryosections for immunostaining. Therefore, the duplicate samples were made available for each assay with six mice per group. Thus, six mice per group were used for each experiment, and each experiment was repeated at least three times. 

Evaluation of Corneal Defects by Oregon Green Dextran Fluorescent Staining

A large molecular size (70 kDa) OGD conjugated with fluorescent dye 488 (Thermo Fisher Scientific, Eugene, OR, USA) was used to assess mouse corneal epithelium defects and permeability using a previously described method.^34,38 The procedure was performed on day 5 in the afternoon, the last day of DS, with instillation of 1.0 µL of OGD (50 mg/mL) into each eye of mice 1 minute before euthanasia. Mice were euthanized by inhalation of isoflurane gas followed by cervical dislocation. The eyes then were rinsed twice with 1 mL of balanced salt solution (BSS). Excess liquid was blotted carefully with filter papers without touching the cornea. Digital pictures of both eyes from each animal were taken for evaluation under 470 nm excitation and 488 nm emission wave lengths using a Nikon SMZ1500 stereo microscope with an exposure time of 200 ms. The mean fluorescence intensity in the central cornea with a diameter of 2 mm was evaluated from digital images using NIS Elements (version 3.0) software (Nikon USA, Melville, NY, USA). Results were presented as geometric mean and standard deviation (SD) of gray levels. 

Measurement of Conjunctival Goblet Cell Density

Eyeballs with ocular adnexa were excised after euthanasia, fixed in 10% formalin, followed by paraffin embedding, and cut into 5 µm sections using a microtome (Microm HM 340E; Thermofisher Wilmington, DE, USA). Sections were stained for goblet cells using a PAS-Periodic/Schiff Stain Kit (K7308; IMEB, San Marcos, CA, USA) and photographed with a microscope (Eclipse E400; Nikon, Melville, NY, USA) equipped with a digital camera (DXM1200; Nikon, Melville, NY, USA). The goblet cell density was measured in the superior or inferior bulbar and tarsal conjunctiva by using NIS-Elements software (AR, version 5.20.2; Nikon, Melville, NY, USA). A line was drawn on the surface of the conjunctiva image from the first to the last PAS-positive goblet cell to determine the length of the conjunctival goblet cell zone. Results were presented as the number of PAS-positive cells per millimeter.³⁹ 

Evaluation of Mucin Production and Expression in Ocular Surface

To evaluate the mucin production by ocular surface, whole eyeballs with ocular adnexa were collected after animal euthanasia. The eyeballs with lids of mice in each group, UT mice, and DS mice topically treated with 2.0% ectoine or PBS vehicle, were excised and embedded in OCT compound (VWR, Suwanee, GA, USA), and flash-frozen in liquid nitrogen. Sagittal cryosections at 8 µm of mouse eyeballs were cut with a cryostat (HM 500; Micron, Waldorf, Germany), and stored at −80°C before being used for immunofluorescent staining. 

To evaluate the mucin mRNA expression by ocular surface, corneal epithelium and conjunctival tissues were excised and pooled from two eyes (right and left) of mice in each group. Each pooled specimen was lysed in 350 µL of RNA lysis buffer (RLT+β-ME, Qiagen, Valencia, CA, USA) and stores at −80°C before being used for total RNA isolation and gene expression analysis. 

Assessment of Cytokines Regulating Mucin Expression

To explore the mechanism by which ectoine enhances ocular mucin production, the expression of Th1 type cytokine IFN-γ and Th2 cytokine IL-13 at mRNA and protein levels was investigated in conjunctival tissue and draining CLNs. These specimens were excised and lysed in RNA lysis buffer for mRNA expression assays, lysed in protein lysis buffer for enzyme-linked immunosorbent assay, or embedded in OCT for cryosections to be used for immunofluorescent staining, respectively. 

Immunofluorescent Staining and Laser Scanning Confocal Microscopy

Immunofluorescent (IF) staining was performed as previously described.^40,41 In brief, the cryosections were thawed, and fixed with cold acetone at −30°C for 3 minutes. After blocking for 60 minutes with 20% normal goat serum in PBS, the specimens were incubated with primary antibodies overnight at 4°C. Primary antibodies used for this study are listed in Table 1. Alexa-Fluor 488 (1:100) was used as the secondary antibody, and DAPI was used for nuclear counterstaining. Secondary antibody alone or isotype IgG was used as negative controls. The digital images were captured with a laser scanning confocal microscope (Nikon A1 RMP; Nikon, Melville, NY, USA) at a wavelength of 400 to 750 nm and one µm z-step. The images were processed using software NIS Elements 4.20 version (Nikon, Garden City, NY, USA). 

Table 1.

 

Antibodies Used in This Study

 

Enzyme-Linked Immunosorbent Assay

Double-sandwich ELISA kits for mouse IFN-γ and IL-13 were obtained from BioLegend (San Diego, CA, USA) and R & D Systems (Minneapolis, MN, USA), respectively. The assays were performed according to manufacturers’ protocols, similar to our previous reports.^42,43 Absorbance was read at 450 nm with a reference wavelength of 570 nm by Infinite M200 microplate reader (Tecan US, Inc., Morrisville, NC, USA). The results were present as pg/mg protein of conjunctival tissue. 

RNA Isolation, Reverse Transcription, and Quantitative Real-Time PCR

Total RNA was isolated from corneal epithelium, conjunctival tissues, and CLNs. In brief, total RNA was extracted using RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions, quantified with a spectrophotometer (NanoDrop ND-1000; Thermo Scientific, Wilmington, DE, USA), and stored at −80°C before use. The first strand cDNA was synthesized by real-time (RT) from total RNA using Ready-To-Go You-Prime First-Strand Beads (GE Healthcare, Piscataway, NJ, USA), as previously described.^44,45 

Quantitative real-time PCR was performed in the QuantStudio 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with 10 µL reaction volume containing 4.5 µL of cDNA, 0.5 µL TaqMan gene expression assay, and 5 µL TaqMan gene expression master mix. The thermocycler parameters were 50°C for 2 minutes and 95°C for 10 minutes, followed by 35 cycles of 95°C for 15 seconds and 60°C for 1 minute. TaqMan gene expression assays used for this study are listed in Table 2. GAPDH (Mm99999915_g1), a non-template control, was included to evaluate DNA contamination. The results were analyzed by the comparative threshold cycle (Ct) method (the 2(-delta delta c(t)) method) and normalized by GAPDH as an internal control.^46–48 

Table 2.

 

Mouse TaqMan Gene Expression Assays Used in This Study

 

Statistical Analysis

Student's t-test or Mann-Whitney U test was used to make comparisons between the two groups. 

One-way ANOVA test was used to make comparisons among three or more groups, followed by Dunnett's post hoc test. P < 0.05 was considered statistically significant. The software GraphPad Prism (version 9; GraphPad Inc.) was used for the statistical analysis. 

Results

Ectoine Protected Corneal Epithelium in a Murine Dry Eye Model

A dry eye model was induced in female C57BL/6 mice exposed to DS for 5 days. Corneal staining with OGD, a large molecular weight fluorescein, was performed on day 5 before euthanization, which has been shown to be a better detection for corneal defects and permeability.^49,50 As shown in Figure 1A, corneal OGD uptake significantly increased with punctate and confluent dye staining in DS mice treated with vehicle when compared with the UT group, indicating the corneal epithelial defect and increased permeability in mice exposed to DS. Whereas OGD uptake by the corneas was dramatically reduced in DS mice topically treated with 2.0% ectoine, suggesting a protective effect of ectoine on corneal epithelium. 

Figure 1.

 

Corneal permeability evaluated by Oregon Green Dextran (OGD) fluorescent staining in mice exposed to desiccating stress (DS). (A) Representative images of corneas stained with 1.0 µL of 50 mg/mL OGD in 3 groups: UT, DS+PBS, and DS+2% ectoine (Ect). (B) The mean fluorescence intensity in the central cornea with a diameter of 2 mm was evaluated after background correction. Each circle represents one eye. Data are shown as Mean ± SD, n = 6; the P values are shown in the graph as compared with the DS+PBS group.

OGD fluorescence intensity quantitatively measured the changes of corneal permeability in three groups of mice (Fig. 1B). Corneal OGD intensity increased to 6.3-fold higher (82.23 ± 24.92, P = 0.0001) in DS mice treated with vehicle than UT controls (13.28 ± 3.31). Topical eyedrops of 2.0% ectoine reduced the fluorescence intensity significantly to 32.63 ± 19.39 (P = 0.0032). 

Ectoine Prevented the Loss of Conjunctival Goblet Cells in Mice Exposed to Desiccating Stress

Conjunctival goblet cell loss has been observed in both patients with Sjögren syndrome and non-Sjogren dry eye,^8,20,21 as well as in murine dry eye models.^22–24 In this study, we detected that the number and size of conjunctival goblet cells that positively stained by PAS with purple magenta color, were largely reduced in DS mice with PBS vehicle when compared with UT controls. Interestingly, 2.0% ectoine eyedrops significantly prevented their loss, the number and size of goblet cells were largely recovered, as shown in Figure 2A. The measurement of goblet cell density quantitatively displayed this pattern of goblet cell number changed in these three groups of mice (Fig. 2B). These results suggest that ectoine topical treatment effectively prevented loss of conjunctival goblet cells, although not completely. 

Figure 2.

 

Ectoine improves goblet cell density in DS mice. (A) Representative images of PAS staining on eyeball sections showing PAS positive conjunctival goblet cells (purple magenta), which largely decreased in the DS+PBS group and significantly increased with the 2% ectoine treatment. Scale bar = 50 µm. (B) Cumulative data of conjunctival goblet cell density in the three groups. Mann–Whitney U test; each circle represents one eye. Data are shown as Mean ± SD, n = 6; the P values are shown in the graph as compared with the DS+PBS group.

Ectoine Enhanced Mucin Production of the Ocular Surface in Murine Dry Eye Model

Mucins play a key role to protect and maintain integrity of the ocular surface. The levels of mucins in the ocular surface and tears have been known to decrease or are altered in patients with dry eye^12,51,52 and animal models.^8,24,53 In this study, we observed that two major secreted mucins, MUC5AC and MUC2, were produced by conjunctival epithelial and goblet cells, but not corneal epithelium. Conjunctival cells produced more abundant MUC5AC than MUC2. However, their production dramatically decreased in DS mice with PBS vehicle, but largely recovered in DS mice treated with 2.0% ectoine, as assessed by IF staining (Figs. 3A, 3C). The IF intensity quantitatively showed the changes of these two secreted mucins in these three groups (Figs. 3B, 3D). 

Figure 3.

 

Ectoine enhances production of secreted mucins in DS mice. (A, C) Representative images of IF staining showing the decreased production of MUC5AC (A) and MUC2 (C) in DS mice (DS+PBS) was recovered by ectoine eye drops (DS+Ect); Scale bars = 50 µm. The stitched images were made because the cornea and conjunctiva in some eyeball sections were too far apart to be captured on the same image. (B, D) IF intensity quantified the alternation of MUC5AC and MUC2 in 3 groups; each circle represents one eye. Data are shown as Mean ± SD, n = 6; the P values are shown in the graph as compared with the DS+PBS group.

We also studied the production of four transmembrane mucins in mouse ocular surface. IF staining showed that MUC1, MUC4, and MUC16 were more abundantly produced by conjunctival epithelium, including goblet cells, than corneal epithelium (Figs. 4A, 4C, 4E); whereas MUC15 was weakly produced by conjunctival epithelium, and not detected in the cornea of normal UT mice (Fig. 4G). The production of these 4 mucins were significantly suppressed in DS mice with PBS vehicle but largely recovered in DS mice treated with 2.0% ectoine (see Figs. 4A, 4C, 4E, 4G). IF intensity quantified the production alterations of these mucins in three groups of mice (Figs. 4B, 4D, 4F, 4H). 

Figure 4.

 

Ectoine enhances production of transmembrane mucins in DS mice. (A, C, E, G) Representative images of IF staining showing the decreased production of MUC1 (A), MUC4 (C), MUC16 (E), and MUC15 (G) in conjunctiva and/or cornea of DS mice (DS+PBS) was recovered by ectoine eye drops (DS+Ect); Scale bars = 50 µm. The stitched images were made because the cornea and conjunctiva in some eyeball sections were too far apart to be captured on the same image. (B, D, F, H) IF intensity quantified the alternation of these four mucins in the three groups; each circle or triangle represents one eye. Data are shown as Mean ± SD, n = 6; the P values are shown in the graph as compared with the DS+PBS group.

Ectoine Upregulated Mucin Expression in Mouse Ocular Surface in the Dry Eye Model

To confirm the changes of these mucin productions, we performed RT-qPCR in UT mice to study their gene expression in normal condition. Figure 5A showed the threshold cycle (Ct) generated by qPCR for each mucin gene with GAPDH as internal control. It is well known that the lower Ct values indicate the higher expression levels, vice versa.^46,54 Consistent with IF staining results, all 6 mucins were expressed by conjunctival cells, which express abundant levels of MUC5AC, MUC1, MUC4, and MUC16 with mean value of Ct cycles that ranged from 19.7 to 23.5, compared with 18.7 of house-keeping gene GAPDH. Conjunctiva expressed lower mRNA levels of MUC2 and MUC15 with Ct values at 30.6 and 27.9, respectively. Corneal epithelium moderately expressed the transcripts of MUC1, MUC4, and MUC16 with Ct cycles that ranged from 20.0 to 23.8, but expressed barely detectable levels of MUC5AC, MUC2, and MUC15 with very higher Ct values (31.7–35.4), which may explain the negative immunoreactivity of these 3 mucins in corneas, as shown in Figures 3A and 3C, and Figure 4G. 

Figure 5.

 

Gene expression of MUC5AC, MUC2, MUC1, MUC4, MUC16 , and MUC15 by conjunctiva and cornea. (A) Gene expression of all 6 mucins in normal mice were detected at different mRNA levels based on the threshold cycle (Ct) value in real-time PCR with house-keeping gene GAPDH as an internal control. (B) The mRNA levels of all six mucins in conjunctiva of the three groups of mice. (C) The mRNA expression of MUC1, MUC4, and MUC16 by corneas in the three groups of mice. Data are shown as Mean ± SD, n = 6; the P values are shown in the graph as compared with the DS+PBS group.

In DS mice exposed to DS, the mRNA expression of all six mucins in conjunctiva was significantly downregulated but largely rebounded in DS mice treated with ectoine, when compared with UT mice as controls (Fig. 5B). The mRNA levels of MUC1, MUC4, and MUC16 in the corneal epithelium were also downregulated in DS mice but recovered with ectoine treatment (Fig. 5C). We did not investigate the mRNA changes of corneal MUC5AC, MUC2, and MUC15 in dry eye mice due to their barely detectable expressions. 

Effects of Ectoine on T Helper Cell Secreted Cytokines in Ocular Surface of Dry Eye Mice

Inflammation is a major driver of pathogenesis and may suppress mucin productions in dry eye disease.^23,55,56 To explore the potential inflammatory cytokines that regulate the mucin production, we assessed the expression of IL-13 and IFN- γ, the respective Th2 and Th1 markers, in conjunctiva that contained the infiltrated immune cells, including T cells in dry eye mice.^57–59 

The protein levels of cytokines in mouse conjunctival tissues were measured by ELISA. Compared with UT mice (513.03 ± 72.58 pg/mg), IL-13 protein production markedly decreased to 350.05 ± 79.14 pg/mg (P = 0.004) in conjunctival tissues of DS mice, whereas they increased to near normal levels (473.84 ± 67.60 pg/mg, P = 0.0155, compared to DS mice with PBS vehicle) by topical administration of 2% ectoine (Fig. 6A). In contrast, compared with UT mice (146.03 ± 21.06 pg/mg), Th1 cytokine IFN-γ production in conjunctiva of DS mice increased to 274.94 ± 77.51 pg/mg (P = 0.0028), whereas significantly decreased to near normal levels (169.63 ± 37.12 pg/mg, P = 0.0133, compared to DS mice with PBS) in DS mice treated with 2% ectoine eye drops (Fig. 6B). The protein production ratio of IL-13/IFN-γ decreased 2.69-fold (P = 0.0001) in DS mice, whereas it significantly recovered to near normal levels (Fig. 6C). 

Figure 6.

 

IL-13 and IFN-γ expression in conjunctiva of 3 groups of mice. (A–C) Protein levels of IL-13, IFN-γ, and IL-13/IFN-γ ratio in conjunctiva were measured by ELISA. (D–F) Their mRNA levels in conjunctiva were evaluated by RT-qPCR. Data are shown as Mean ± SD, n = 6; the P values are shown in the graph as compared with the DS+PBS group.

The mRNA levels of conjunctival IL-13 and IFN-γ by RT-qPCR showed a consistent expression pattern to protein levels detected by ELISA. IL-13 mRNA expression was dramatically suppressed, whereas IFN-γ mRNA was markedly stimulated in DS mice, but the treatment of 2% ectoine reversed the changes of their expression (Figs. 6D, 6E). The mRNA expression ratio of IL-13/IFN-γ significantly decreased 3.70-fold (P = 0.0001) in DS mice, whereas it recovered to near normal levels (Fig. 6F), the pattern was similar to protein levels. 

Effects of Ectoine on Th Cytokines IL-13/IFN-γ in Ocular Surface Draining CLNs

Inflammation in the ocular surface can induce innate and/or adaptive immune responses, which can be detected in conjunctiva with infiltrated immune cells, as well as in ocular draining CLNs. The pathological responses of various immune cells, including dendritic cells, macrophages, and T cells, have been observed in our previous studies on ocular allergic disease^45,60,61 and dry eye^50,62 in mouse models. 

Interestingly, IF staining on CLN cryosections showed that IL-13 positive cells largely decreased in DS mice, whereas it significantly increased to near normal levels by topical treatment of 2% ectoine (Fig. 7A). In contrast, compared with UT mice, IFN-γ positive cells increased significantly in CLNs of DS mice but decreased to near normal levels in DS mice treated with 2% ectoine eye drops (Fig. 7C). Consistently, the mRNA levels of IL-13 and IFN-γ showed a similar expression pattern to IF staining results. IL-13 mRNA expression in CLNs of DS mice was dramatically downregulated to 41% of the levels in normal UT mice, but it recovered to 94% of normal expression levels in DS mice treated with 2% ectoine (Fig. 7B). In contrast, IFN-γ mRNA was also markedly upregulated to 2.24-fold in DS mice, but downregulated to near normal levels by ectoine treatment (Fig. 7D). 

Figure 7.

 

IL-13 and IFN-γ expression in draining cervical lymph nodes (CLNs). (A, C) Representative images of IF staining on CLN cryosections showing that the decreased production of IL-13 (A) and increased IFN-γ (C) in DS mice (DS+PBS) was recovered by ectoine eye drops (DS+Ect); Scale bars = 50 µm. (B, D) IF intensity quantified the alternation of IL-13 and IFN-γ in the 3 groups. Data are shown as Mean ± SD, n = 6; the P values are shown in the graph as compared with the DS+PBS group.

Discussion

The lacrimal functional unit, which is consisted of the cornea, conjunctiva, lacrimal glands, meibomian glands, and its associated nerves, produces the tear film that lubricates and protects the ocular surface from environmental insults.^5,8,63 Mucins, a large family of heavily glycosylated proteins, are a crucial component of the tear film and play a key role in maintaining integrity of the healthy ocular surface.^4,7,9 Dry eye disease is a multifactorial disorder, which affects up to 30% of the US population.⁶⁴ Ocular mucins play a vital role in the pathogenesis of dry eye disease. The reduced production of glycocalyx mucins and loss of goblet cells have been recognized as major hallmarks of dry eye disease.⁶⁵ However, the beneficial role of osmoprotectant solution in protecting ocular surface mucins has not been reported. The present study aimed to explore the protective effects and potential mechanism of ectoine on ocular mucin expression in dry eye disease using a murine model exposed to DS.^36,66 

The symptoms of patients with dry eye are mainly derived from ocular surface epithelial defect due to chronic desiccation. We assessed corneal epithelial defect using OGD fluorescent staining in our murine dry eye model. Although corneal epithelial defects are usually examined by staining with 0.1% liquid sodium fluorescein clinically, OGD, a large size of 70 kDa fluorescein, can better detect corneal epithelial defects and permeability in a murine dry eye model.⁴⁹ A previous study showed that 10 kDa Dextran bound to the epithelium surface but migrated in a columnar pattern into the stroma while 70 kDa Dextran primarily stained to the epithelium surface.⁵⁰ In this study, corneal uptake of OGD significantly increased with punctate and confluent dye staining in DS mice with PBS vehicle (see Fig. 1), like human dry eye, where sodium fluorescein is used to detect the increased corneal epithelial permeability.⁶⁷ The fluorescein intensity of OGD staining quantitatively showed the similar patterns to corneal OGD uptake. Interestingly, corneal OGD uptake was significantly reduced to near normal levels in DS mice treated with 2.0% of ectoine eye drops, suggesting its protective effects on corneal epithelium. 

Goblet cells are a major source of mucins,⁵⁷ and loss of conjunctival goblet cells is a hallmark observed in patients with Sjögren syndrome dry eye.^8,21,68 In this mouse dry eye model, with conjunctival goblet cell density and the cell size largely decreased, indicating not only goblet cell number, but also its function of producing mucins were markedly reduced. Interestingly, topical ectoine eye drops significantly restored goblet cell density and size (see Fig. 2), suggesting that ectoine may prevent goblet cell loss in DS mice exposed to DS. 

Conjunctival goblet cell loss often results in decrease of mucin production or mucin deficiency in dry eye syndrome.^20,69 MUC5AC and MUC2 are major gel-forming mucins secreted by conjunctiva and detected in tears,⁷⁰ but not expressed by the corneas. MUC5AC is abundantly produced in major amounts by goblet cells,⁷¹ and known as a hallmark for human patients with dry eye^72,73 and animal dry eye models.^25,74 MUC2 is expressed by conjunctival epithelial cells at low levels,^4,75 but MUC2 in dry eye is less investigated, although a few studies reported the decreased mRNA levels of MUC 2 gene expression in patients with dry eye.¹² In this study, we confirmed that conjunctival MUC5AC and MUC2 were significantly suppressed at both the protein and mRNA levels by IF staining and RT-qPCR, respectively, in a murine dry eye model (see Figs. 3, 5). 

Transmembrane mucins, the distinguishing components of the mucosal glycocalyx, maintain ocular surface wetness, stabilize the tear film, lubricate the blink, create a physical barrier, and serve as cell surface receptors transducing signals.^4,76 MUC1, MUC4, and/or MUC16 were reported to be expressed by human corneal and conjunctival epithelia,^16,77,78 and their expression and/or production were reduced in dry eye disease, including Sjögren syndrome, visual display terminal users, and contact lens wear.^12,13,72,79 However, most of these reports showed expression at mRNA levels, not confirmed at protein levels. MUC15 is known to play a crucial role in malignant tumors and serve as a tumor suppressor.⁸⁰ MUC15 expression in the ocular surface is not clear and there is no report on its role in dry eye disease. In this study, we investigated the expression of these four transmembrane mucins at both protein and mRNA levels in ocular surface and their alternation in the mouse dry eye model. 

Our results confirmed that MUC1, MUC4, and MUC16 were abundantly expressed in conjunctival epithelium, whereas weakly by corneal epithelium. In contrast, MUC15 was only expressed at low levels by conjunctival but not corneal epithelium. We further observed that the expressions of these four mucins at the mRNA and protein levels were significantly suppressed in mice exposed to DS (see Figs. 4, 5). 

Mucin expression in responses to different treatment in dry eye disease has been investigated. For example, rebamipide eye drops, a mucin secretagogue, was reported to increase MUC5AC and MUC4 levels in tear film and reduce the severity of GVHD-corneal dry eye.^72,81 Topical cyclosporine A (CsA) and diquafosol tetrasodium (DQS) not only ameliorated ocular surface inflammation but also upregulated MUC4, MUC16, and MUC5AC, and promote goblet cell recovery in a dry eye mice model.²⁴ Osmoprotectants have been known to suppress the inflammatory markers and relieve the severity of dry eye conditions.⁸² However, their effects on mucin deficiency in dry eye have not been reported. In this study, we explored whether ectoine, a potent osmoprotectant, can promote mucin production in a dry eye model. Our results demonstrated that ectoine not only enhances the secreted MUC5AC and MUC2, but also upregulated transmembrane mucins MUC1, MUC4, MUC16, and MUC15 at both the protein and mRNA levels (see Figs. 3–5). 

The abnormality of mucins expression in dry eye may be attributed to ocular surface inflammation, which is a major driver of pathogenesis in dry eye disease. We have observed that ectoine suppressed the proinflammatory cytokines and chemokines in dry eye mice exposed to DS.³⁴ This study explored new findings. We observed that the protein and mRNA expression of Th2 cytokine IL-13, evaluated by ELISA and RT-qPCR, was significantly inhibited, whereas Th1 cytokine IFN-γ levels were largely increased in ocular surface and its draining CLNs, leading to decreased ratio of IL-13 over IFN-γ (see Figs. 6, 7). Interestingly, ectoine eye drops reversed these alterations by enhancing IL-13 and suppressing IFN-γ, leading to rebalanced ratio of IL-13/IFN-γ. IL-13 has been recognized to stimulate proliferation and expression of mucins, such as MUC5AC and MUC2, in conjunctival goblet cells.⁵⁷ In contrast, IFN-γ is known to suppress mucin production, including MUC5AC and MUC2,²⁰ so that the imbalance ratio of IL-13/IFN-γ caused mucin deficiency. These findings suggest that ectoine prevents ocular surface from mucin deficiency by rebalancing Th cell signaling, the ratio of IL-13/IFN-γ expression. However, further studies are necessary to clarify how ectoine promotes mucins through IL-13/IFN-γ signaling pathway. In addition, other pathways may be involved and need to be further investigated. 

In conclusion, corneal epithelial defects, conjunctival goblet cell loss, and ocular surface mucin deficiency were observed in a mouse model of dry eye disease. Furthermore, Th2 cytokine IL-13 expression was inhibited, whereas Th1 cytokine IFN-γ level was stimulated in conjunctiva and draining CLNs of DS mice, Interestingly, topical ectoine treatment reversed these alternations and restored IL-13/IFN-γ balance. These findings demonstrate that ectoine possesses therapeutic potential to reduce corneal damage, increase goblet cell density, and enhance mucin production through restoring imbalanced IL-13/IFN-γ signaling in murine dry eye model. This provides new insight into pathogenesis and therapeutic potential for dry eye disease. 

Acknowledgments

Supported in part by the National Institutes of Health Grants R01 EY023598 (D.Q.L.), EY011915 (S.C.P.), and the Core Grant for Vision Research EY002520 (Bethesda, DC, USA), AbbVie Inc. (Irvine, CA, USA), National Natural Science Foundation of China (82000858, X.C.), Natural Science Foundation of Zhejiang Province (LQ20H120005, X.C.), the Research to Prevent Blindness (New York, NY, USA). 

Disclosure: N. Lin, None; X. Chen, None; H. Liu, (F); N. Gao, None; Z. Liu, None; J. Li, None; S.C. Pflugfelder, (F); D.-Q. Li, (F)