September 2017
Volume 58, Issue 11
Open Access
Cornea  |   September 2017
Therapeutic Effect of MK2 Inhibitor on Experimental Murine Dry Eye
Author Affiliations & Notes
  • Yang Wu
    Eye Institute of Xiamen University and Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Jinghua Bu
    Eye Institute of Xiamen University and Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Yiran Yang
    Eye Institute of Xiamen University and Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Xiang Lin
    Eye Institute of Xiamen University and Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Xiaoxin Cai
    Eye Institute of Xiamen University and Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Caihong Huang
    Eye Institute of Xiamen University and Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Xiaoxiang Zheng
    Eye Institute of Xiamen University and Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Weijie Ouyang
    Eye Institute of Xiamen University and Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Wei Li
    Eye Institute of Xiamen University and Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Xiaobo Zhang
    Eye Institute of Xiamen University and Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Zuguo Liu
    Eye Institute of Xiamen University and Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Correspondence: Zuguo Liu, Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiangan South Road, Xiamen, Fujian, China; zuguoliu@xmu.edu.cn
  • Xiaobo Zhang, Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiangan South Road, Xiamen, Fujian, China; xiaoboz@xmu.edu.cn
  • Footnotes
     YW and JB contributed equally to the work present here and should therefore be regarded as equivalent authors.
  • Footnotes
     ZL and XZ contributed equally to the work present here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science September 2017, Vol.58, 4898-4907. doi:10.1167/iovs.17-22240
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Yang Wu, Jinghua Bu, Yiran Yang, Xiang Lin, Xiaoxin Cai, Caihong Huang, Xiaoxiang Zheng, Weijie Ouyang, Wei Li, Xiaobo Zhang, Zuguo Liu; Therapeutic Effect of MK2 Inhibitor on Experimental Murine Dry Eye. Invest. Ophthalmol. Vis. Sci. 2017;58(11):4898-4907. doi: 10.1167/iovs.17-22240.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: To investigate the role of mitogen-activated protein kinase–activated protein kinase-2 (MK2) in ocular surface damage of dry eye.

Methods: MK2 inhibition was performed in mice subjected to desiccating stress (DS) by topical application of MK2 inhibitor (MK2i) or vehicle eye drops. The total and phosphorylated MK2 in conjunctiva were detected by Western blot. The phenol red cotton test was used to measure tear production, and Oregon green dextran staining was performed to assess corneal epithelial barrier function. PAS staining was used to quantify conjunctival goblet cells. Immunofluorescent staining and quantitative RT-PCR were used to assess the expression of matrix metalloproteinase (MMP)-3 and -9 in corneal epithelium. Apoptosis in ocular surface was assessed by TUNEL and immunofluorescent staining for activated caspase-3 and -8. Inflammation was evaluated by CD4+ T-cell infiltration and production of T helper (Th) cytokines, including IFN-γ, IL-13, and IL-17A in conjunctiva.

Results: DS promoted MK2 activation in conjunctiva. Compared with vehicle control mice, MK2i-treated mice showed increased tear production, decreased goblet cell loss, and improved corneal barrier function. Topical MK2 inhibition decreased the expression of MMP-3 and -9 in corneal epithelium, and suppressed cell apoptosis in ocular surface under DS. Topical MK2 inhibition decreased CD4+ T-cell infiltration, with decreased production of IFN-γ and IL-17A and increased production of IL-13 in conjunctiva.

Conclusions: Topical MK2 inhibition effectively alleviated ocular surface damage via suppressing cell apoptosis and CD4+ T-cell–mediated inflammation in ocular surface of dry eye.

Dry eye is a frequent ocular disease affecting 10% to 30% of the population all over the world.1 Dry eye is known as a multifactorial disease of the tear fluid and ocular surface that results in symptoms of discomfort, visual disturbance, tear film instability, and potential damage to the ocular surface. Dry eye is accompanied by increased osmolality of the tear film and inflammation in the ocular surface.2 Dry eye may cause various problems with the eyes, including dryness, redness, foreign body sensation, tearing, and photophobia. Dry eye seriously affects the patient's work efficiency and quality of life.13 
It is well known that dry eye is an inflammatory ocular surface disease. Chronic inflammation stimulated by the activation of innate immune in the ocular surface, instability of the tear film, and the hyperosmolar tears plays a vital role in the immuno-pathogenic mechanism of dry eye.2,46 Whatever the initial etiology of dry eye, once it has developed, inflammation becomes the key mechanism of ocular surface damage, as both the cause and consequence of cell apoptosis and squamous metaplasia.2,46 It has been well documented that treatment aimed at suppressing the ocular surface inflammation could effectively improve tear film stability, normalize tear osmolality, sustain ocular surface homeostasis, and alleviate ocular discomfort.7 
Mitogen-activated protein kinase–activated protein kinase-2 (MK2) is a member of the serine/threonine protein kinase family that is activated by direct phosphorylation by p38. MK2 is a key participant in the inflammation-related signaling pathways. It has been confirmed that MK2 is crucial for lipopolysaccharide (LPS)-induced upregulation of cytokine mRNA stability and translation that regulates the biosynthesis of inflammatory cytokines, such as proinflammatory cytokines (TNF-α and IL-6), chemokines (IL-8), and adhesion molecules (vascular cell adhesion molecule-1).812 MK2 activation plays a pivotal role in cell response to inflammation in various diseases, including tumor necrosis, pancreatitis, and postoperative ileus.1315 Previously, we have found that MK2 plays a key role in alkali burn-induced corneal inflammation.16 Topical inhibition of MK2 activation suppressed immune cell infiltration, stromal thickening, and declines of corneal epithelial and endothelial integrity in alkali burned corneas.16 MK2 inhibitor (MK2i) may be a suitable compound to reduce inflammation in various ocular surface diseases. Until now, the role of MK2 in the ocular surface inflammation of dry eye remains unknown. 
The purpose of this study was to investigate the role of MK2 activation in the ocular surface inflammation of dry eye. 
Materials and Methods
Mice
The protocol of this research was approved by the Experimental Animal Ethics Committee of Xiamen University and it conformed to the standards in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Female C57BL/6 (B6) mice, aged 6 to 8 weeks, were purchased from Shanghai SLAC Laboratory Animal Center, Shanghai, China. 
Murine Dye Eye Model
Desiccating stress (DS) was used to induce experimental dry eye in B6 mice by subcutaneous injection of scopolamine hydrobromide (0.5 mg/0.2 mL; catalog no. MB5860; Melonepharma, Dalian, China) four times a day (9:00, 12:00, 15:00, 18:00) and exposure to an air draft and less than 40% ambient humidity for 5 days. A group of age- and gender-matched mice that did not receive any treatment to induce dry eye served as nonstressed (NS) controls. 
Topical MK2i Treatment in B6 Mice Subjected to DS
Topical MK2i treatment was performed in B6 mice subjected to DS by topical application of 5 μL MK2i (DS5+MK2i; 12.5 μg/mL, 25 μg/mL, or 50 μg/mL; catalog no. 475864; Millipore, Billerica, MA, USA) or vehicle (PBS containing 1/40,000, 1/20,000, or 1/10,000 DMSO; DS5+Vehicle) eye drops four times daily for 5 days under DS. 
Western Blot
The conjunctivae of both eyes of one mouse were collected and pooled as one sample, and five samples were used in each group. The conjunctiva was then minced and lysed in cold radioimmunoprecipitation assay (RIPA) buffer containing a proteinase inhibitor cocktail (catalog no. 78440; ThermoFisher Scientific, Waltham, MA, USA). The total protein concentration of the supernatant was measured with a BCA protein assay kit (catalog no. 23225; ThermoFisher Scientific). Aliquots having equal protein content were subjected to electrophoresis on 10% Tricine gels and then electronically transferred to PVDF membranes (catalog no. IPVH00010; Millipore, Billerica, MA, USA). After blocking in 5% BSA for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies for MK2 (1:500; catalog no. ab131531; Abcam, Cambridge, UK), Phospho-MK2 (1:500; catalog no. ab63378; Abcam) and horseradish peroxidase (HRP)-conjugated anti-β-actin antibody (1:20,000; catalog no. a5316; Sigma-Aldrich Corp., St. Louis, MO, USA). After washing three times with Tris-buffered saline containing 0.05% Tween 20 for 10 minutes, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:10,000; catalog no. a0545; Sigma-Aldrich Corp.) for 1 hour at room temperature. The specific bands were visualized by an enhanced chemiluminescence reagent (catalog no. ECL-500; ECL, Lulong, Inc., Xiamen, China), and the image intensity was calculated with a transilluminator (ChemiDoc XRS System; Bio-Rad, Philadelphia, PA, USA). 
Measurement of Tear Production
Tear production (10 eyes/five mice per group) was measured with phenol red–impregnated cotton threads (Zone-Quick; Yokota, Tokyo, Japan) at the same time point (8 PM). The thread was placed on the lower conjunctival fornix at approximately one-third of the lower eyelid distance from the lateral canthus for 15 seconds. The length of the wet red thread was measured in millimeters. 
Corneal Permeability
Corneal epithelial permeability to Oregon green dextran (OGD) (70,000 molecular weight; catalog no. D7172; Invitrogen, Eugene, OR, USA) was assessed (10 eyes/five mice per group) as previously described.17 Briefly, OGD (0.5 μL of 50 mg/mL) was instilled onto the ocular surface 1 minute before killing. Corneas were rinsed five times with saline and then photographed with a stereoscopic zoom microscope (AZ100; Nikon, Tokyo, Japan) under fluorescence excitation at 470 nm. The mean intensity of corneal OGD staining was measured in digital images using the analysis software (NIS Elements, version 4.1; Nikon, Melville, NY, USA). A 2-mm diameter circle was placed on the central cornea, and the mean fluorescent intensity in this circle was measured by the software. 
Histology
The eyes and adnexa of mice were excised, embedded in optimal cutting temperature (OCT) compound (catalog no.4583; SAKURA Tissue-Tek, Torrance, CA, USA) or paraffin. OCT-embedded samples were cut into sagittal sections (6 μm thick), and then placed onto glass slides that were stored at −80°C. PAS staining and TUNEL assay were performed on paraffin sections (5 μm thick), and immunostaining was performed on frozen sections (two sections per slide, three slides per animal, five animals per group). 
PAS Staining
The mucin-filled goblet cells (GCs) in the conjunctiva were stained using a PAS staining kit (catalog no. 395B-1KT; Sigma-Aldrich Corp.). Digital images of representative areas of the conjunctiva were captured with the light microscope (Eclipse 50i; Nikon, Tokyo, Japan). 
Immunofluorescent Staining
Immunofluorescent staining was performed in cryosections of the eyes and adnexa. Sections were fixed in acetone at −20°C, and then incubated at 4°C overnight with polyclonal goat anti–matrix metalloproteinase (MMP)-3 antibody (1:50; catalog no. sc-6839; Santa Cruz Biotechnology, Dallas, TX, USA), goat anti-MMP-9 antibody (1:50; catalog no. sc-6840; Santa Cruz Biotechnology), rabbit anti-activated (AC) caspase-3 antibody (1:250; catalog no. ab52181; Abcam), or rabbit anti-AC-caspase-8 antibody (1:50; catalog no. sc-7890; Santa Cruz Biotechnology). Negative controls were performed at the same time by incubating a section with just PBS without any primary antibody. The next day, samples were incubated with Alexa Fluor488-conjugated donkey anti-goat (1:300; catalog no. A11055; Invitrogen, Eugene, OR, USA) or anti-rabbit IgG (1:300; catalog no. A21206; Invitrogen) for 1 hour in the dark at room temperature, followed by three washes in PBS. Sections were then counterstained with 4′,6-diamidino-2-phenylindole (DAPI; catalog no. H-1200; Vector, Burlingame, CA, USA) for 5 minutes. Digital images of representative areas of the cornea or conjunctiva were captured with the Leica upright microscope (DM2500; Leica Microsystems, Wetzlar, Germany). The mean intensity of staining in each section was measured by analysis software (NIS Elements version 4.1; Nikon, Melville, NY, USA). 
TUNEL Assay
To measure the end-stage apoptosis, in situ TUNEL assay (DeadEnd Fluorometric TUNEL System; catalog no. G3250; Promega, Madison, WI, USA) was performed on paraffin sections according to the manufacturer's instructions. Sections were counterstained with DAPI (catalog no. H-1200; Vector), and digital images of representative areas of the cornea and conjunctiva were captured with the Leica upright microscope (DM2500; Leica Microsystems). 
Immunohistochemistry
Immunohistochemistry was performed to detect and count the cells in the conjunctiva that stained positively for CD4. Cryosections were stained with primary rat anti-mouse CD4 antibody (1:50; catalog no. 553647; BD Pharmingen, San Diego, CA, USA), goat anti-rat antibody (1:25; catalog no. 559286; BD Pharmingen) and Vectastain Elite ABC using NovaRed reagents (catalog no. PK-6100; Vector). Secondary antibody alone and appropriate anti-mouse isotype (BD Pharmingen) controls were also performed. Three slides from each animal were examined and photographed with a microscope equipped with a digital camera (Eclipse 50i; Nikon, Tokyo, Japan). Positively stained cells were counted in the conjunctiva using image analysis software (NIS Elements, version 4.1, Nikon, Melville, NY, USA). 
RNA Extraction and Quantitative RT-PCR
Murine corneal epithelium was scraped with a scalpel, conjunctiva was surgically excised, and total RNA was isolated from the corneal epithelium or conjunctiva by a PicoPure RNA isolation kit (catalog no. KIT0204; Arcturus, Mountain View, CA, USA). Five samples per group were used, and one sample consisted of pooled corneal epithelium or conjunctiva of both eyes of the same animal. cDNA was synthesized using a reverse transcription kit (catalog no. RR047A; TaKaRa, Shiga, Japan). Real-time PCR was performed on StepOne Real-Time PCR System (Applied Biosystems, Alameda, CA, USA) using a SYBR Premix Ex TaqKit (catalog no. RR420A; TaKaRa), and the primer sequences are summarized in the Table. The amplification program included an initial denaturation step at 95°C for 10 minutes, followed by 40 cycles of 95°C for 10 seconds, and 60°C for 30 seconds, after which a melt curve analysis was conducted to check amplification specificity. The results of real-time PCR were analyzed by the comparative cycle threshold (Ct) method where target change = 2 − ΔΔCt, normalized with β-actin as an endogenous reference, and calibrated against the NS group. 
Table
 
Mouse Primer Sequences Used for Quantitative RT-PCR
Table
 
Mouse Primer Sequences Used for Quantitative RT-PCR
Mouse IL-17A, IL-13, and IFN-γ ELISA Assay
The conjunctiva proteins of each group (five samples per group, and one sample consisted of pooled conjunctiva of both eyes of the same animal) were extracted with cold RIPA buffer (catalog no. R0278; Sigma-Aldrich Corp.), and total protein concentration of the cell extract was measured with a BCA protein assay kit (catalog no. 23225; ThermoFisher Scientific). The same dilution ratio was used in all groups. ELISA kits were used to detect the protein concentrations of IL-17A (catalog no. BMS6001; eBioscience, San Diego, CA, USA), IL-13 (catalog no. BMS6015; eBioscience), and IFN-γ (catalog no. BMS606; eBioscience) in conjunctiva according to manufacturer's instructions. The optical absorbance was measured at 450 nm with a microplate reader (Bio TekElx800; Bio-Tek Instruments, Winooski, VT, USA), and the protein concentrations were calculated according to the standard curve. 
Statistical Analysis
One-way ANOVA with Tukey's post hoc test was conducted for statistical comparison between groups using GraphPad Prism 5.0 software (GraphPad Software, Inc, San Diego, CA, USA). P ≤ 0.05 was considered statistically significant. 
Results
The Activation of MK2 in Ocular Surface During DS
As shown in Figure 1, DS promoted MK2 activation in the conjunctiva, and topical application of MK2i eye drops suppressed the activation of MK2 in the conjunctiva. In this study, 25 μg/mL was chosen as the adopted concentration to assess the effect of MK2i on ocular surface damage in dry eye induced by DS. 
Figure 1
 
Effects of topical application of MK2i on DS-induced MK2 activation in the conjunctiva. MK2 activation was evaluated by Western blot, with β-actin as a loading control. The level of phosphorylated-MK2 (p-MK2) (A) and total-MK2 (B) in conjunctiva. (C) The ratio of the intensity of p-MK2 to total MK2 in conjunctiva. Data shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 1
 
Effects of topical application of MK2i on DS-induced MK2 activation in the conjunctiva. MK2 activation was evaluated by Western blot, with β-actin as a loading control. The level of phosphorylated-MK2 (p-MK2) (A) and total-MK2 (B) in conjunctiva. (C) The ratio of the intensity of p-MK2 to total MK2 in conjunctiva. Data shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
Topical Application of MK2i Alleviated DS-Induced Ocular Surface Damage
Topical application of MK2i eye drops improved tear production (Fig. 2A), increased the number of conjunctival GCs (Figs. 2B, 2C), and alleviated the corneal barrier dysfunction shown by OGD staining (Figs. 2D, 2E) in experimental dry eye. 
Figure 2
 
Effects of topical application of MK2i on DS-induced ocular surface damage.
 
(A) Phenol red cotton test for quantification of tear production. (B) Representative images of PAS staining in conjunctiva. (C) The total number of GCs in conjunctiva. (D) Representative images of OGD staining in cornea. (E) The mean intensity of corneal OGD staining. Data shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 100 μm for PAS staining, and 500 μm for OGD staining.
Figure 2
 
Effects of topical application of MK2i on DS-induced ocular surface damage.
 
(A) Phenol red cotton test for quantification of tear production. (B) Representative images of PAS staining in conjunctiva. (C) The total number of GCs in conjunctiva. (D) Representative images of OGD staining in cornea. (E) The mean intensity of corneal OGD staining. Data shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 100 μm for PAS staining, and 500 μm for OGD staining.
Topical Application of MK2i Decreased the Expression of MMP-9 and MMP-3 in Corneal Epithelium During DS
Previous studies have shown that disruption of the corneal barrier in dry eye is associated with increased production of MMPs, particularly MMP-9 and MMP-3.1720 This study investigated the expression of MMP-9 and MMP-3 in corneal epithelium using immunofluorescent staining and quantitative RT-PCR, and found that topical application of MK2i eye drops suppressed the expression of MMP-9 and MMP-3 in corneal epithelium during DS (Fig. 3). 
Figure 3
 
Effects of topical application of MK2i on expression of MMPs in corneal epithelium during DS. (A) Representative merged images of MMP-3 (green) immunofluorescent staining in corneal epithelium with DAPI counterstaining (blue) in nucleus. (B) The immunofluorescence intensity of MMP-3 in corneal epithelium. (C) The mRNA levels of MMP-3 in corneal epithelium. (D) Representative merged images of MMP-9 (green) immunofluorescent staining in corneal epithelium with DAPI counterstaining (blue) in nucleus. (E) The immunofluorescence intensity of MMP-9 in corneal epithelium. (F) The mRNA levels of MMP-9 in corneal epithelium. Data shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Figure 3
 
Effects of topical application of MK2i on expression of MMPs in corneal epithelium during DS. (A) Representative merged images of MMP-3 (green) immunofluorescent staining in corneal epithelium with DAPI counterstaining (blue) in nucleus. (B) The immunofluorescence intensity of MMP-3 in corneal epithelium. (C) The mRNA levels of MMP-3 in corneal epithelium. (D) Representative merged images of MMP-9 (green) immunofluorescent staining in corneal epithelium with DAPI counterstaining (blue) in nucleus. (E) The immunofluorescence intensity of MMP-9 in corneal epithelium. (F) The mRNA levels of MMP-9 in corneal epithelium. Data shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Topical Application of MK2i Suppressed DS-induced Apoptosis in Ocular Surface
A growing body of clinical and experimental studies has shown that pathologic apoptosis has a key role in the pathogenesis of dry eye disease.2123 In this study, the number of TUNEL-positive cells was decreased in both the corneal epithelium and conjunctiva in the MK2i-treated group compared with the vehicle control group (Fig. 4). In addition, this study found that topical application of MK2i eye drops suppressed the immunoreactivity of AC-Caspase-3 and AC-Caspase-8 in ocular surface under DS (Figs. 5, 6). 
Figure 4
 
Effects of topical application of MK2i on DS-induced cell apoptosis on ocular surface. (A) Representative images for TUNEL staining in corneal epithelium and conjunctiva; The number of TUNEL-positive cells in corneal epithelium (B) and conjunctiva (C). Data was shown as mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Figure 4
 
Effects of topical application of MK2i on DS-induced cell apoptosis on ocular surface. (A) Representative images for TUNEL staining in corneal epithelium and conjunctiva; The number of TUNEL-positive cells in corneal epithelium (B) and conjunctiva (C). Data was shown as mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Figure 5
 
Effects of topical application of MK2i on expression of AC-Caspase-3 on ocular surface. (A) Representative merged images of AC-Caspase-3 (green) immunofluorescent staining in corneal epithelium and conjunctiva with DAPI counterstaining (blue) in nucleus; The immunofluorescence intensity of AC-Caspase-3 in corneal epithelium (B) and conjunctiva (C). Data was shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Figure 5
 
Effects of topical application of MK2i on expression of AC-Caspase-3 on ocular surface. (A) Representative merged images of AC-Caspase-3 (green) immunofluorescent staining in corneal epithelium and conjunctiva with DAPI counterstaining (blue) in nucleus; The immunofluorescence intensity of AC-Caspase-3 in corneal epithelium (B) and conjunctiva (C). Data was shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Figure 6
 
Effects of topical application of MK2i on expression of AC-Caspase-8 on ocular surface. (A) Representative merged images of AC-Caspase-8 (green) immunofluorescent staining in corneal epithelium and conjunctiva with DAPI counterstaining (blue) in nucleus; The immunofluorescence intensity of AC-Caspase-8 in corneal epithelium (B) and conjunctiva (C). Data was shown as mean ± SD. **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Figure 6
 
Effects of topical application of MK2i on expression of AC-Caspase-8 on ocular surface. (A) Representative merged images of AC-Caspase-8 (green) immunofluorescent staining in corneal epithelium and conjunctiva with DAPI counterstaining (blue) in nucleus; The immunofluorescence intensity of AC-Caspase-8 in corneal epithelium (B) and conjunctiva (C). Data was shown as mean ± SD. **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Topical Application of MK2i Suppressed CD4+ T-Cell–Mediated Inflammation in Conjunctiva During DS
Dry eye is an inflammatory ocular surface disease characterized by infiltration of CD4+ T cells producing IFN-γ and IL-17A in conjunctiva.17,24,25 The results of this study indicated that topical application of MK2i effectively suppressed the DS-induced infiltration of CD4+ T cells in conjunctiva (Figs. 7A, 7B). Topical application of MK2i decreased the production of IL-17A and IFN-γ and increased the production of IL-13 in conjunctiva under DS (Figs. 7C, 7D). 
Figure 7
 
Effects of topical application of MK2i on DS-induced inflammation in conjunctiva. (A) Representative images of immunohistochemical staining for CD4+ T cells in conjunctiva; (B) The number of CD4+ T cells in conjunctiva; (C) The mRNA levels of IFN-γ, IL-13 and IL-17A in conjunctiva; (D) The protein levels of IFN-γ, IL-13 and IL-17A in conjunctiva detected by ELISA assay. Data was shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Figure 7
 
Effects of topical application of MK2i on DS-induced inflammation in conjunctiva. (A) Representative images of immunohistochemical staining for CD4+ T cells in conjunctiva; (B) The number of CD4+ T cells in conjunctiva; (C) The mRNA levels of IFN-γ, IL-13 and IL-17A in conjunctiva; (D) The protein levels of IFN-γ, IL-13 and IL-17A in conjunctiva detected by ELISA assay. Data was shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Discussion
MK2 is a kinase that is exclusively activated on stress by p38. The activation of MK2 can be stimulated in various inflammatory conditions, and MK2-induced production of proinflammatory cytokine has been observed in several inflammatory conditions.8,10,12 It has been confirmed that MK2 activation plays a crucial role in a vast amount of functions linked with inflammation and may thus be a potential drug target for inflammatory diseases, such as arthritis, atherosclerosis, and cancer.26 In this study, we present evidence that topical application of MK2i eye drops could effectively alleviate epithelial damage, reduce cell apoptosis, and suppress CD4+ T-cell–mediated inflammation in the ocular surface in an experimental murine model of dry eye. 
Dry eye is a chronic inflammatory disease characterized by infiltration of CD4+ T cells producing IFN-γ and IL-17A in conjunctiva.17,24,25 Adoptive transfer of CD4+ T cells isolated from mice subjected to DS to the nude mice induces dry eye–like ocular surface damage in the nude mice, suggesting that CD4+ T-cell–mediated inflammation plays a vital role in the pathogenesis of dry eye.24 The pathogenetic mechanisms responsible for CD4+ T-cell–induced ocular surface damage in dry eye are not completely understood; however, there is increasing evidence indicating that an altered balance of Th cytokines are capable of altering ocular surface epithelial homeostasis. It has been reported that inflammatory cytokines (IFN-γ and IL-17A) released by the resident intraepithelial lymphocytes and infiltrating CD4+ T cells are obviously increased, whereas the production of Th2 cytokine IL-13, which has a homeostatic function in maintaining conjunctival GCs, is notably decreased in the ocular surface of murine dry eye.17,25,2729 This study investigated the role of MK2 activation in ocular surface inflammation during DS, and found that topical application of MK2i significantly alleviated DS-induced inflammation in conjunctiva by suppressing infiltration of CD4+ T cells and production of IFN-γ and IL-17A, suggesting that MK2 activation has a crucial role in CD4+ T-cell–mediated inflammation in the ocular surface of dry eye. 
It is well recognized that cell apoptosis on the ocular surface has a key role in the pathogenesis of dry eye disease, and apoptosis has been defined as a therapeutic target for dry eye.2123,3032 Apoptosis occurs through two pathways: the extrinsic pathway mediated by the binding of death ligands (e.g., TNF-α, Fas) with their corresponding cell surface receptors and the activation of caspase-8; and the intrinsic pathway mediated by DNA damage and subsequent activation of caspase-9. Both pathways result in downstream activation of effector caspases, such as caspase-3.3335 Previously, it has been reported that DS induces conjunctival apoptosis mainly through the caspase-8–mediated extrinsic pathway.23,31 This study found that topical application of MK2i notably suppressed the immune-reactivity of AC-Caspase-3 and -8, and reduced the number of TUNEL-positive cells in ocular surface under DS, suggesting that topical application of MK2i could suppress the DS-induced cell apoptosis via extrinsic apoptosis pathway in ocular surface. 
Disruption of corneal epithelial barrier function is a distinct clinical feature of dry eye disease. Corneal barrier dysfunction can lead to ocular irritation, ocular surface irregularity, blurred vision, and visual morbidity.36,37 MMP-9 has been found to have a central role in DS-induced corneal barrier disruption, as MMP-9–deficient mice were resistant to barrier disruption in DS.1820 It has been reported that IL-17 neutralization during DS decreased the expression of MMP-9 and MMP-3 and improved corneal barrier function.17,28 MMP-3 is known to be the main activator of MMP-9. In the present study, topical application of MK2i decreased corneal OGD staining and expression of IL-17A, MMP-3, and MMP-9 on the ocular surface, indicating that topical application of MK2i may rescue corneal barrier function via suppression of the expression of IL-17A, MMP-3, and MMP-9 on the ocular surface in experimental dry eye. 
Conjunctival GCs are highly secretory epithelial cells that secrete the gel-forming mucin, MUC5AC, that lubricates the ocular surface and stabilizes the tear film. It has been confirmed that the imbalance between Th1 and Th2 cytokines plays an essential role in conjunctival GC loss during DS. Th1 and Th2 cytokines have been found to have opposing effects on conjunctival GC development. IL-13, the predominant Th2 cytokine, appears to have a homeostatic function in promoting GC differentiation.38 In contrast, the Th1 cytokine IFN-γ can promote GC loss via inducing apoptosis and suppressing IL-13 signaling.39 This study showed that topical application of MK2i notably increased the number of conjunctival GCs during DS, and this is accompanied by increased production of IL-13 and decreased production of IFN-γ in conjunctiva, suggesting topical application of MK2i may rescue the GC loss via modulating the balance between IL-13 and IFN-γ. 
Dry eye is a chronic inflammatory disease with frequent recurrence and needs long-term treatment. It has been confirmed that no obvious side effect was noted after systemic application of MK2i for 4 weeks in vivo.40 Previously, we found that topical application of MK2i for 7 days has no obvious side effect on the ocular surface; however, the safety issue for long-term application MK2i eye drops on the ocular surface has not yet been studied. Future research is necessary to investigate the safety of long-term use of MK2i eye drops on the ocular surface. 
In conclusion, this study provides evidence that topical application of MK2i could effectively alleviate ocular surface damage via suppressing cell apoptosis and CD4+ T-cell–mediated inflammation on the ocular surface in an experimental murine dry eye model. This study provides new insight for the pathogenesis of dry eye, and MK2 is a potential therapeutic target of dry eye. 
Acknowledgments
Supported by grants from National Key Scientific Research Project (No. 2013CB967003 to ZL), and the National Natural Science Foundation of China (No. 81500693 to XZ, and No. 81330022 to ZL). The authors alone are responsible for the writing and the content of the paper. 
Disclosure: Y. Wu, None; J. Bu, None; Y. Yang, None; X. Lin, None; X. Cai, None; C. Huang, None; X. Zheng, None; W. Ouyang, None; W. Li, None; X. Zhang, None; Z. Liu, None 
References
Hong S, Kim T, Chung SH, Kim EK, Seo KY. Recurrence after topical nonpreserved methylprednisolone therapy for keratoconjunctivitis sicca in Sjogren's syndrome. J Ocul Pharmacol Ther. 2007; 23: 78–82.
The definition and classification of dry eye disease: report of the Definition and Classification Subcommittee of the International Dry Eye WorkShop (2007). Ocul Surf. 2007; 5: 75–92.
Rapoport Y, Singer JM, Ling JD, Gregory A, Kohanim S. A comprehensive review of sex disparities in symptoms, pathophysiology, and epidemiology of dry eye syndrome. Semin Ophthalmol. 2016; 31: 325–336.
Barabino S, Chen Y, Chauhan S, Dana R. Ocular surface immunity: homeostatic mechanisms and their disruption in dry eye disease. Prog Retin Eye Res. 2012; 31: 271–285.
Stevenson W, Chauhan SK, Dana R. Dry eye disease: an immune-mediated ocular surface disorder. Arch Ophthalmol. 2012; 130: 90–100.
Baudouin C, Aragona P, Messmer EM, et al. Role of hyperosmolarity in the pathogenesis and management of dry eye disease: proceedings of the OCEAN group meeting. Ocul Surf. 2013; 11: 246–258.
Management and therapy of dry eye disease: report of the Management and Therapy Subcommittee of the International Dry Eye WorkShop (2007). Ocul Surf. 2007; 5: 163–178.
Kotlyarov A, Neininger A, Schubert C, et al. MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis. Nat Cell Biol. 1999; 1: 94–97.
Winzen R, Kracht M, Ritter B, et al. The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism. EMBO J. 1999; 18: 4969–4980.
Gorska MM, Liang Q, Stafford SJ, et al. MK2 controls the level of negative feedback in the NF-kappaB pathway and is essential for vascular permeability and airway inflammation. J Exp Med. 2007; 204: 1637–1652.
Mourey RJ, Burnette BL, Brustkern SJ, et al. A benzothiophene inhibitor of mitogen-activated protein kinase-activated protein kinase 2 inhibits tumor necrosis factor alpha production and has oral anti-inflammatory efficacy in acute and chronic models of inflammation. J Pharmacol Exp Ther. 2010; 333: 797–807.
Neininger A, Kontoyiannis D, Kotlyarov A, et al. MK2 targets AU-rich elements and regulates biosynthesis of tumor necrosis factor and interleukin-6 independently at different post-transcriptional levels. J Biol Chem. 2002; 277: 3065–3068.
Ray AL, Castillo EF, Morris KT, et al. Blockade of MK2 is protective in inflammation-associated colorectal cancer development. Int J Cancer. 2016; 138: 770–775.
Liu X, Wu T, Chi P. Inhibition of MK2 shows promise for preventing postoperative ileus in mice. J Surg Res. 2013; 185: 102–112.
Tietz AB, Malo A, Diebold J, et al. Gene deletion of MK2 inhibits TNF-alpha and IL-6 and protects against cerulein-induced pancreatitis. Am J Physiol Gastrointest Liver Physiol. 2006; 290: G1298–G1306.
Chen Y, Yang W, Zhang X, et al. MK2 inhibitor reduces alkali burn-induced inflammation in rat cornea. Sci Rep. 2016; 6: 28145.
De Paiva CS, Chotikavanich S, Pangelinan SB, et al. IL-17 disrupts corneal barrier following desiccating stress. Mucosal Immunol. 2009; 2: 243–253.
De Paiva CS, Corrales RM, Villarreal AL, et al. Corticosteroid and doxycycline suppress MMP-9 and inflammatory cytokine expression, MAPK activation in the corneal epithelium in experimental dry eye. Exp Eye Res. 2006; 83: 526–535.
Corrales RM, Stern ME, De Paiva CS, Welch J, Li DQ, Pflugfelder SC. Desiccating stress stimulates expression of matrix metalloproteinases by the corneal epithelium. Invest Ophthalmol Vis Sci. 2006; 47: 3293–3302.
Pflugfelder SC, Farley W, Luo L, et al. Matrix metalloproteinase-9 knockout confers resistance to corneal epithelial barrier disruption in experimental dry eye. Am J Pathol. 2005; 166: 61–71.
Brignole F, De Saint-Jean M, Goldschild M, Becquet F, Goguel A, Baudouin C. Expression of Fas-Fas ligand antigens and apoptotic marker APO2.7 by the human conjunctival epithelium. Positive correlation with class II HLA DR expression in inflammatory ocular surface disorders. Exp Eye Res. 1998; 67: 687–697.
Rybickova I, Vesela V, Fales I, Skalicka P, Jirsova K. Apoptosis of conjunctival epithelial cells before and after the application of autologous serum eye drops in severe dry eye disease. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2016; 160: 271–275.
Yeh S, Song XJ, Farley W, Li D-Q, Stern ME, Pflugfelder SC. Apoptosis of ocular surface cells in experimentally induced dry eye. Invest Opthalmol Vis Sci. 2003; 44: 124–129.
Niederkorn JY, Stern ME, Pflugfelder SC, et al. Desiccating stress induces T cell-mediated Sjogren's syndrome-like lacrimal keratoconjunctivitis. J Immunol. 2006; 176: 3950–3957.
Yoon KC, De Paiva CS, Qi H, et al. Expression of Th-1 chemokines and chemokine receptors on the ocular surface of C57BL/6 mice: effects of desiccating stress. Invest Ophthalmol Vis Sci. 2007; 48: 2561–2569.
Duraisamy S, Bajpai M, Bughani U, Dastidar SG, Ray A, Chopra P. MK2: a novel molecular target for anti-inflammatory therapy. Expert Opin Ther Targets. 2008; 12: 921–936.
Zhang X, De Paiva CS, Su Z, Volpe EA, Li DQ, Pflugfelder SC. Topical interferon-gamma neutralization prevents conjunctival goblet cell loss in experimental murine dry eye. Exp Eye Res. 2014; 118: 117–124.
Zhang X, Volpe EA, Gandhi NB, et al. NK cells promote Th-17 mediated corneal barrier disruption in dry eye. PLoS One. 2012; 7: e36822.
Zheng X, de Paiva CS, Li DQ, Farley WJ, Pflugfelder SC. Desiccating stress promotion of Th17 differentiation by ocular surface tissues through a dendritic cell-mediated pathway. Invest Ophthalmol Vis Sci. 2010; 51: 3083–3091.
Brignole F, Pisella PJ, De Saint Jean M, Goldschild M, Goguel A, Baudouin C. Flow cytometric analysis of inflammatory markers in KCS: 6-month treatment with topical cyclosporin A. Invest Ophthalmol Vis Sci. 2001; 42: 90–95.
Zhang X, Chen W, De Paiva CS, et al. Interferon-gamma exacerbates dry eye-induced apoptosis in conjunctiva through dual apoptotic pathways. Invest Ophthalmol Vis Sci. 2011; 52: 6279–6285.
Strong B, Farley W, Stern ME, Pflugfelder SC. Topical cyclosporine inhibits conjunctival epithelial apoptosis in experimental murine keratoconjunctivitis sicca. Cornea. 2005; 24: 80–85.
Nunez G, Benedict MA, Hu Y, Inohara N. Caspases: the proteases of the apoptotic pathway. Oncogene. 1998; 17: 3237–3245.
Barrett KL, Willingham JM, Garvin AJ, Willingham MC. Advances in cytochemical methods for detection of apoptosis. J Histochem Cytochem. 2001; 49: 821–832.
Chawla-Sarkar M, Lindner DJ, Liu YF, et al. Apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis. Apoptosis. 2003; 8: 237–249.
Gobbels M, Spitznas M. Corneal epithelial permeability of dry eyes before and after treatment with artificial tears. Ophthalmology. 1992; 99: 873–878.
Yokoi N, Kinoshita S. Clinical evaluation of corneal epithelial barrier function with the slit-lamp fluorophotometer. Cornea. 1995; 14: 485–489.
De Paiva CS, Raince JK, McClellan AJ, et al. Homeostatic control of conjunctival mucosal goblet cells by NKT-derived IL-13. Mucosal Immunol. 2011; 4: 397–408.
De Paiva CS, Villarreal AL, Corrales RM, et al. Dry eye-induced conjunctival epithelial squamous metaplasia is modulated by interferon-gamma. Invest Ophthalmol Vis Sci. 2007; 48: 2553–2560.
Song H, Fang X, Wen M, et al. Role of MK2 signaling pathway in the chronic compression of cervical spinal cord. Am J Transl Res. 2015; 7: 2355–2363.
Figure 1
 
Effects of topical application of MK2i on DS-induced MK2 activation in the conjunctiva. MK2 activation was evaluated by Western blot, with β-actin as a loading control. The level of phosphorylated-MK2 (p-MK2) (A) and total-MK2 (B) in conjunctiva. (C) The ratio of the intensity of p-MK2 to total MK2 in conjunctiva. Data shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 1
 
Effects of topical application of MK2i on DS-induced MK2 activation in the conjunctiva. MK2 activation was evaluated by Western blot, with β-actin as a loading control. The level of phosphorylated-MK2 (p-MK2) (A) and total-MK2 (B) in conjunctiva. (C) The ratio of the intensity of p-MK2 to total MK2 in conjunctiva. Data shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 2
 
Effects of topical application of MK2i on DS-induced ocular surface damage.
 
(A) Phenol red cotton test for quantification of tear production. (B) Representative images of PAS staining in conjunctiva. (C) The total number of GCs in conjunctiva. (D) Representative images of OGD staining in cornea. (E) The mean intensity of corneal OGD staining. Data shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 100 μm for PAS staining, and 500 μm for OGD staining.
Figure 2
 
Effects of topical application of MK2i on DS-induced ocular surface damage.
 
(A) Phenol red cotton test for quantification of tear production. (B) Representative images of PAS staining in conjunctiva. (C) The total number of GCs in conjunctiva. (D) Representative images of OGD staining in cornea. (E) The mean intensity of corneal OGD staining. Data shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 100 μm for PAS staining, and 500 μm for OGD staining.
Figure 3
 
Effects of topical application of MK2i on expression of MMPs in corneal epithelium during DS. (A) Representative merged images of MMP-3 (green) immunofluorescent staining in corneal epithelium with DAPI counterstaining (blue) in nucleus. (B) The immunofluorescence intensity of MMP-3 in corneal epithelium. (C) The mRNA levels of MMP-3 in corneal epithelium. (D) Representative merged images of MMP-9 (green) immunofluorescent staining in corneal epithelium with DAPI counterstaining (blue) in nucleus. (E) The immunofluorescence intensity of MMP-9 in corneal epithelium. (F) The mRNA levels of MMP-9 in corneal epithelium. Data shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Figure 3
 
Effects of topical application of MK2i on expression of MMPs in corneal epithelium during DS. (A) Representative merged images of MMP-3 (green) immunofluorescent staining in corneal epithelium with DAPI counterstaining (blue) in nucleus. (B) The immunofluorescence intensity of MMP-3 in corneal epithelium. (C) The mRNA levels of MMP-3 in corneal epithelium. (D) Representative merged images of MMP-9 (green) immunofluorescent staining in corneal epithelium with DAPI counterstaining (blue) in nucleus. (E) The immunofluorescence intensity of MMP-9 in corneal epithelium. (F) The mRNA levels of MMP-9 in corneal epithelium. Data shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Figure 4
 
Effects of topical application of MK2i on DS-induced cell apoptosis on ocular surface. (A) Representative images for TUNEL staining in corneal epithelium and conjunctiva; The number of TUNEL-positive cells in corneal epithelium (B) and conjunctiva (C). Data was shown as mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Figure 4
 
Effects of topical application of MK2i on DS-induced cell apoptosis on ocular surface. (A) Representative images for TUNEL staining in corneal epithelium and conjunctiva; The number of TUNEL-positive cells in corneal epithelium (B) and conjunctiva (C). Data was shown as mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Figure 5
 
Effects of topical application of MK2i on expression of AC-Caspase-3 on ocular surface. (A) Representative merged images of AC-Caspase-3 (green) immunofluorescent staining in corneal epithelium and conjunctiva with DAPI counterstaining (blue) in nucleus; The immunofluorescence intensity of AC-Caspase-3 in corneal epithelium (B) and conjunctiva (C). Data was shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Figure 5
 
Effects of topical application of MK2i on expression of AC-Caspase-3 on ocular surface. (A) Representative merged images of AC-Caspase-3 (green) immunofluorescent staining in corneal epithelium and conjunctiva with DAPI counterstaining (blue) in nucleus; The immunofluorescence intensity of AC-Caspase-3 in corneal epithelium (B) and conjunctiva (C). Data was shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Figure 6
 
Effects of topical application of MK2i on expression of AC-Caspase-8 on ocular surface. (A) Representative merged images of AC-Caspase-8 (green) immunofluorescent staining in corneal epithelium and conjunctiva with DAPI counterstaining (blue) in nucleus; The immunofluorescence intensity of AC-Caspase-8 in corneal epithelium (B) and conjunctiva (C). Data was shown as mean ± SD. **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Figure 6
 
Effects of topical application of MK2i on expression of AC-Caspase-8 on ocular surface. (A) Representative merged images of AC-Caspase-8 (green) immunofluorescent staining in corneal epithelium and conjunctiva with DAPI counterstaining (blue) in nucleus; The immunofluorescence intensity of AC-Caspase-8 in corneal epithelium (B) and conjunctiva (C). Data was shown as mean ± SD. **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Figure 7
 
Effects of topical application of MK2i on DS-induced inflammation in conjunctiva. (A) Representative images of immunohistochemical staining for CD4+ T cells in conjunctiva; (B) The number of CD4+ T cells in conjunctiva; (C) The mRNA levels of IFN-γ, IL-13 and IL-17A in conjunctiva; (D) The protein levels of IFN-γ, IL-13 and IL-17A in conjunctiva detected by ELISA assay. Data was shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Figure 7
 
Effects of topical application of MK2i on DS-induced inflammation in conjunctiva. (A) Representative images of immunohistochemical staining for CD4+ T cells in conjunctiva; (B) The number of CD4+ T cells in conjunctiva; (C) The mRNA levels of IFN-γ, IL-13 and IL-17A in conjunctiva; (D) The protein levels of IFN-γ, IL-13 and IL-17A in conjunctiva detected by ELISA assay. Data was shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 50 μm.
Table
 
Mouse Primer Sequences Used for Quantitative RT-PCR
Table
 
Mouse Primer Sequences Used for Quantitative RT-PCR
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×