January 2013
Volume 54, Issue 1
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Immunology and Microbiology  |   January 2013
Therapeutic Effect of Topical Adiponectin in a Mouse Model of Desiccating Stress–Induced Dry Eye
Author Affiliations & Notes
  • Zhengri Li
    From the Department of Ophthalmology and Research Institute of Medical Sciences, Chonnam National University Medical School and Hospital, Gwangju, Korea; the
  • Je Moon Woo
    Department of Ophthalmology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Korea; and the
  • Su Wol Chung
    School of Biological Sciences, Ulsan University, Ulsan, Korea.
  • Min-Young Kwon
    School of Biological Sciences, Ulsan University, Ulsan, Korea.
  • Ji-Suk Choi
    From the Department of Ophthalmology and Research Institute of Medical Sciences, Chonnam National University Medical School and Hospital, Gwangju, Korea; the
  • Han-Jin Oh
    From the Department of Ophthalmology and Research Institute of Medical Sciences, Chonnam National University Medical School and Hospital, Gwangju, Korea; the
  • Kyung-Chul Yoon
    From the Department of Ophthalmology and Research Institute of Medical Sciences, Chonnam National University Medical School and Hospital, Gwangju, Korea; the
  • Corresponding author: Kyung-Chul Yoon, Department of Ophthalmology, Chonnam National University Medical School and Hospital, 8 Hak-Dong, Dong-Gu, Gwangju 501-757, South Korea; kcyoon@jnu.ac.kr
Investigative Ophthalmology & Visual Science January 2013, Vol.54, 155-162. doi:10.1167/iovs.12-10648
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      Zhengri Li, Je Moon Woo, Su Wol Chung, Min-Young Kwon, Ji-Suk Choi, Han-Jin Oh, Kyung-Chul Yoon; Therapeutic Effect of Topical Adiponectin in a Mouse Model of Desiccating Stress–Induced Dry Eye. Invest. Ophthalmol. Vis. Sci. 2013;54(1):155-162. doi: 10.1167/iovs.12-10648.

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

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Abstract

Purpose.: To investigate the therapeutic effect of topical adiponectin in a mouse model of experimental dry eye (EDE).

Methods.: EDE was created by desiccating stress in 6- to 8-week old female C57BL/6 mice. Eye drops consisting of 0.0001%, 0.001%, or 0.01% adiponectin, or balanced salt solution (BSS), were applied in EDE. Tear volume and corneal irregularity score were measured at 5 and 10 days after treatment. Levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and monokine induced by interferon-γ (MIG) were measured in the conjunctiva and lacrimal gland using a multiplex immunobead assay at 10 days. Periodic acid–Schiff staining, immunohistochemistry, and flow cytometry were also performed.

Results.: Mice treated with 0.001% or 0.01% adiponectin showed a significant improvement in tear volume and corneal irregularity compared with the EDE control and BSS-treated groups. A significant decrease in the levels of IL-1β, IL-6, TNF-α, IFN-γ, and MIG and staining intensity of TNF-α was observed in the 0.001% and 0.01% adiponectin-treated groups, compared with the other groups, in the conjunctiva and lacrimal gland. In the 0.001% and 0.01% adiponectin-treated groups, the density of conjunctival goblet cells was higher and the number of CD4+CXCR3+ T cells was lower than in the other groups. However, there were no significant differences in all parameters between the 0.0001% adiponectin and control groups.

Conclusions.: Topical application of adiponectin can markedly improve clinical signs and decrease inflammation in the ocular surface and lacrimal gland of EDE, suggesting that adiponectin eye drops may be used as a therapeutic agent for dry eye disease.

Introduction
Dry eye disease is a chronic and progressive condition of the ocular surface that affects tens of millions of patients worldwide. 1 Recently, it has become widely recognized that inflammation plays a key role in the pathogenesis of dry eye disease. Known inflammatory changes in the ocular surface of dry eye are increased expression of immune activation and apoptosis markers, adhesion molecules, matrix metalloproteinases, inflammatory cytokines, chemokines and their receptors, and CD4+ T cells. 26  
The management of dry eye disease includes environmental modification, artificial tears, anti-inflammatory agents, punctal plugs, serum eye drops, contact lenses, and surgery according to disease severity. 7,8 Recent treatments of dry eye have been focused on the inhibition of inflammation in the lacrimal functional unit. Topical anti-inflammatory agents such as corticosteroids and cyclosporin A improve symptoms, as well as tear film and ocular surface parameters, by inhibiting inflammation in the ocular surface and lacrimal gland. 913  
Adiponectin is a 244 amino acid long polypeptide protein secreted mainly by the adipose tissue. 14 This 30 kDa protein is structurally similar to tumor necrosis factor (TNF)-α and has many pleiotropic effects consisting of antidiabetic, antiatherogenic, and antiangiogenic actions. 1517 Accumulating evidence suggests that adiponectin also exerts a potent immunoregulatory effect, as evidenced by increased levels of the anti-inflammatory cytokines interleukin (IL)-10 and IL-1 receptor antagonist (IL-1RA) and decreased levels of proinflammatory IL-6, TNF-α, and interferon (IFN)-γ. 1820 In addition, adiponectin can reduce the phagocytic activity of macrophages and inhibit the production of CXC receptor 3 (CXCR3) ligands in macrophages. 21  
The involvement and clinical importance of adiponectin have been studied in several systemic and local immune and inflammatory diseases, such as systemic lupus erythematosus, Sjögren's syndrome (SS), and rheumatoid arthritis (RA). 2224 In the eyes, expression of adiponectin and its receptors has been identified in choroidal tissue of mice, and systemic administration of adiponectin was shown to inhibit laser-induced choroidal neovascularization in a mouse model. 25 Recently, it was also demonstrated that adiponectin, as an immunoregulatory hormone, was expressed by ductal epithelial cells in minor salivary glands of patients with primary SS as well as controls. 23 However, no study has been performed on the role of adiponectin in the field of ocular surface inflammatory disorders, including dry eye disease. In this study, we evaluated the therapeutic effect of topical adiponectin for the treatment of dry eye disease, using a desiccating stress–induced mouse model, by evaluating the changes of tear production, ocular surface irregularities, inflammatory cytokines, and T cells on the ocular surface and lacrimal gland, as well as conjunctival goblet cell density. 
Materials and Methods
Mouse Model of Dry Eye and Experimental Procedure
This research protocol was approved by the Chonnam National University Medical School Research Institutional Animal Care and Use Committee. All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Six- to eight-week-old female C57BL/6 mice were used in these experiments. Experimental dry eye (EDE) was induced by subcutaneous injection of 0.5 mg/0.2 mL scopolamine hydrobromide (Sigma-Aldrich, St. Louis, MO) four times a day (8 AM, 11 AM, 2 PM, and 5 PM) with exposure to an air draft and 30% ambient humidity, as previously described. 2629 During these experiments, the animals' behavior, food, and water intake were not restricted. 
The mice were randomly assigned to six groups according to topical treatment administered as follows: (1) untreated (UT) control mice that were not exposed to desiccating stress or treated topically; (2) EDE control mice that received no eye drops; (3) EDE mice treated with balanced salt solution (BSS; Alcon, Fort Worth, TX), (4) EDE mice treated with 0.0001% adiponectin; (5) EDE mice treated with 0.001% adiponectin; and (6) EDE mice treated with 0.01% adiponectin. Adiponectin eye drops were made by diluting globular monomeric fragment recombinant mouse gAdiponectin/gAcro30 solution (R&D Systems, Minneapolis, MN) with BSS. All treatment groups received 2 μL eye drops four times a day. Tear volume and corneal smoothness were measured at 5 and 10 days after treatment. Ten days after treatment, the mice were euthanized, and multiplex immunobead assay, histology, immunohistochemistry, and flow cytometry were performed. Each group consisted of five animals, and the experiments were performed on four independent sets of mice. 
Measurement of Tear Volume
Tear volume was measured using phenol red–impregnated cotton threads (Zone-Quick; Oasis, Glendora, CA), as previously described. 30 The threads were placed in the lateral canthus for 20 seconds. The distances of threads wet by tears were measured using the SMZ 1500 microscope (Nikon, Tokyo, Japan). A standard curve was derived to convert distance into volume. 
Evaluation of Corneal Surface Irregularity
Severity of corneal surface irregularity was graded via measurement of the distortion of a white ring from the fiberoptic ring illuminator of the stereoscopic zoom microscope (SMZ 1500; Nikon) by two masked observers. The corneal irregularity severity score was calculated using a 6-point scale (0–5) based on the number of distorted quarters in the reflected ring, as follows: 0, no distortion; 1, distortion in one quarter of the ring; 2, distortion in two quarters; 3, distortion in three quarters; 4, distortion in all four quadrants; 5, severe distortion, in which no ring could be recognized. 31  
Multiplex Immunobead Assay
A multiplex immunobead assay (Luminex 200; Luminex Corp., Austin, TX) was used to measure the concentrations of IL-1β, IL-6, TNF-α, IFN-γ, and monokine induced by interferon-γ (MIG) in the conjunctiva and lacrimal gland, as previously described. 32 The tissues were collected and pooled in lysis buffer containing protease inhibitors for 30 minutes. The cell extracts were centrifuged at 14,000g for 15 minutes at 4°C, and the supernatants were stored at −70°C before use. The supernatants were added to wells containing the appropriate cytokine bead mixture that included mouse monoclonal antibodies specific for IL-1β, IL-6, TNF-α, IFN-γ, and MIG for 60 minutes. After three washes with assay buffer, the biotinylated secondary cytokine antibody mixture was applied for 30 minutes in the dark at room temperature. The reactions were detected after addition of streptavidin-phycoerythrin with an analysis system (xPONENT, Austin, TX). The concentrations of these factors in tissue were calculated from standard curves of known concentrations of recombinant mouse cytokines. 
Histology and Immunohistochemistry
Eye and adnexa were surgically excised, fixed in 4% paraformaldehyde, and embedded in paraffin. Six-micrometer sections were stained with periodic acid–Schiff (PAS) reagent. Sections were examined and photographed with a microscope (BX53; Olympus, Tokyo, Japan) equipped with a digital camera (F2; Foculus, Finning, Germany). Goblet cell density in the superior and inferior conjunctiva was measured in three sections from each eye using image analysis software (Media Cybernetics, Silver Spring, MD) and expressed as the number of goblet cells per 100 μm. 
Immunohistochemistry was performed to detect the expression of adiponectin receptors, AdipoR1 and AdipoR2, in the conjunctiva of normal eyes and TNF-α in the conjunctiva and lacrimal gland of experimental dry eyes. Hydrogen peroxide (H2O2, 0.3%) in phosphate-buffered saline (PBS) and 20% serum in PBS were sequentially applied to the sections. Conjunctival sections from UT control mice were incubated with goat anti-adiponectin receptor AdipoR1 and AdipoR2 antibodies (Vector Laboratories, Burlingame, CA). Conjunctival and lacrimal gland sections from mice with EDE were incubated with goat monoclonal anti-mouse TNF-α antibody (Santa Cruz Biotechnology, Santa Cruz, CA). After washing, appropriate secondary antibodies were applied. The samples were incubated with avidin-peroxidase, then incubated with 3,3′-diaminobenzidine peroxidase substrate and counterstained with Mayer's hematoxylin. 
Flow Cytometry
Flow cytometry was performed for quantitation of CD4+CXCR3+ T cells from the conjunctiva and lacrimal gland with a previously described method. 6,33 The tissues were teased and shaken at 37°C for 60 minutes with 0.5 mg/mL collagenase type D. After grinding with a syringe plunder and passage through a cell strainer, cells were obtained, centrifuged, and resuspended in PBS with 1% bovine serum albumin. After washing, the samples were incubated with fluorescein-conjugated anti-CD4 antibody (BD Biosciences, San Jose, CA), phycoerythrin-conjugated anti-CXCR3 antibody (BD Biosciences), and isotype control antibody at 37°C for 30 minutes. The number of CD4+CXCR3+ T cells was counted by a FACSCalibur cytometer with CellQuest software (BD Biosciences). 
Statistical Analysis
Statistical differences in the tear volume and corneal irregularity score results were evaluated by one-way ANOVA, with post hoc analysis. Kruskal-Wallis and Mann-Whitney test were used to compare the cytokine level, goblet cell density, and flow cytometry between groups. A P value < 0.05 was considered statistically significant. 
Results
Aqueous Tear Production
Five days after induction of EDE, the mean tear volume was significantly reduced in the EDE group (0.016 ± 0.008 μL) compared with the UT control group (0.034 ± 0.009 μL; P < 0.01). The mean tear volumes at 5 days were 0.021 ± 0.011 μL in the BSS-treated group (P = 0.41 compared with the EDE control), 0.022 ± 0.012 μL in the 0.0001% adiponectin-treated group (P = 0.25 compared with the EDE control; P = 0.47 compared with the BSS-treated group), 0.028 ± 0.009 μL in the 0.001% adiponectin-treated group (P < 0.05 compared with the EDE control or BSS-treated group), and 0.031 ± 0.011 μL in the 0.01% adiponectin-treated group (P < 0.05 compared with the EDE control or BSS-treated group). The findings for mean volumes in all groups at 10 days after treatment were similar to those at 5 days (Fig. 1). 
Figure 1. 
 
Mean tear volumes in the untreated (UT) control, experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated groups at days 5 and 10. *P < 0.05, **P < 0.01 compared with the EDE group. †P < 0.05, ††P < 0.01 compared with the BSS group.
Figure 1. 
 
Mean tear volumes in the untreated (UT) control, experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated groups at days 5 and 10. *P < 0.05, **P < 0.01 compared with the EDE group. †P < 0.05, ††P < 0.01 compared with the BSS group.
Corneal Surface Irregularities
Five days after treatment, the mean corneal irregularity severity score significantly increased in the EDE group (3.78 ± 0.59) compared with the UT control group (0.43 ± 0.11; P < 0.01). The mean scores at 5 days after treatment were 2.97 ± 0.35 in the BSS group (P = 0.63 compared with the EDE control), 2.64 ± 0.29 in the 0.0001% adiponectin-treated group (P = 0.42 compared with the EDE control; P = 0.30 compared with the BSS-treated group), 1.32 ± 0.19 in the 0.001% adiponectin group (P < 0.05 compared with the EDE control or BSS group), and 1.12 ± 0.15 in the 0.01% adiponectin group (P < 0.05 compared with the EDE control or BSS group). The findings for mean scores in all groups at 10 days after treatment were similar to those at 5 days (Figs. 2A, 2B). 
Figure 2. 
 
Mean corneal smoothness scores (A) and representative figures (B) in the untreated (UT) control, experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated groups at days 5 and 10. *P < 0.05, **P < 0.01 compared with the EDE group. †P < 0.05, ††P < 0.01 compared with the BSS group.
Figure 2. 
 
Mean corneal smoothness scores (A) and representative figures (B) in the untreated (UT) control, experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated groups at days 5 and 10. *P < 0.05, **P < 0.01 compared with the EDE group. †P < 0.05, ††P < 0.01 compared with the BSS group.
Inflammatory Cytokine Levels in Conjunctival Tissues and Lacrimal Glands
The results of inflammatory cytokine levels in conjunctival tissues and lacrimal glands are presented in Tables 1 and 2. The concentrations of IL-1β, IL-6, TNF-α, IFN-γ, and MIG in the conjunctiva and lacrimal gland of the EDE control and BSS-treated groups were significantly higher than those of the UT group (P < 0.05). There were no significant differences in the concentrations between the EDE and BSS groups. Conjunctival and lacrimal gland IL-1β, IL-6, TNF-α, IFN-γ, and MIG concentrations in the 0.001% and 0.01% adiponectin-treated groups were significantly lower compared with those in the EDE or BSS group (P < 0.05). However, the 0.0001% adiponectin-treated group did not show significant differences in the concentrations when compared with the EDE control or BSS-treated group. 
Table 1. 
 
Concentrations of IL-1β, IL-6, TNF-α, INF-γ, and MIG in the Conjunctiva of UT Control, EDE Control, BSS-Treated, 0.0001% Adiponectin-Treated, 0.001% Adiponectin-Treated, and 0.01% Adiponectin-Treated Groups
Table 1. 
 
Concentrations of IL-1β, IL-6, TNF-α, INF-γ, and MIG in the Conjunctiva of UT Control, EDE Control, BSS-Treated, 0.0001% Adiponectin-Treated, 0.001% Adiponectin-Treated, and 0.01% Adiponectin-Treated Groups
IL-1β, pg/mL IL-6, pg/mL TNF-α, pg/mL INF-γ, pg/mL MIG, μg/mL
UT 1.08 ± 0.40†§ 0.90 ± 0.19†‡ 0.50 ± 0.18*§ 25.88 ± 3.87†§ 1.99 ± 0.43†§
EDE 6.82 ± 1.01 2.95 ± 0.85 1.59 ± 0.39 47.93 ± 6.41 4.84 ± 0.88
BSS 7.29 ± 1.62 2.59 ± 0.39 1.38 ± 0.41 42.46 ± 5.91 4.77 ± 0.86
0.0001% adiponectin 6.81 ± 1.00 2.57 ± 0.43 1.21 ± 0.21 43.45 ± 3.03 4.25 ± 0.49
0.001% adiponectin 1.30 ± 0.33*‡ 1.02 ± 0.27*‡ 0.55 ± 0.24*‡ 31.95 ± 3.27*‡ 2.20 ± 0.50*‡
0.01% adiponectin 1.22 ± 0.31*‡ 0.99 ± 0.33*‡ 0.52 ± 0.30*‡ 31.44 ± 2.88*‡ 2.17 ± 0.46*‡
Table 2. 
 
Concentrations of IL-1β, IL-6, TNF-α, INF-γ, and MIG in the Lacrimal Gland of UT Control, EDE Control, BSS-Treated, 0.0001% Adiponectin-Treated, 0.001% Adiponectin-Treated, and 0.01% Adiponectin-Treated Groups
Table 2. 
 
Concentrations of IL-1β, IL-6, TNF-α, INF-γ, and MIG in the Lacrimal Gland of UT Control, EDE Control, BSS-Treated, 0.0001% Adiponectin-Treated, 0.001% Adiponectin-Treated, and 0.01% Adiponectin-Treated Groups
IL-1β, pg/mL IL-6, pg/mL TNF-α, pg/mL INF-γ, pg/mL MIG, μg/mL
UT 26.47 ± 3.98†§ 30.73 ± 5.21†§ 0.79 ± 0.26†§ 83.43 ± 11.31*‡ 40.98 ± 6.21†§
EDE 47.98 ± 5.12 70.37 ± 7.23 3.99 ± 0.72 151.21 ± 20.23 90.45 ± 10.06
BSS 45.31 ± 4.31 60.11 ± 9.21 3.51 ± 0.78 132.37 ± 19.08 75.32 ± 9.23
0.0001% adiponectin 40.53 ± 2.09 59.79 ± 4.61 3.61 ± 0.45 137.45 ± 10.33 70.11 ± 6.20
0.001% adiponectin 33.23 ± 4.23*‡ 36.13 ± 5.16*‡ 0.83 ± 0.20*‡ 94.62 ± 10.20*‡ 48.76 ± 7.13*‡
0.01% adiponectin 31.32 ± 4.50*‡ 34.42 ± 4.28*‡ 0.81 ± 0.22*‡ 89.57 ± 11.48*‡ 42.19 ± 6.23*‡
Histology and Immunohistochemistry
The mean conjunctival goblet cell density significantly decreased in the EDE group (16.1 ± 3.3 cells/100 μm) compared with the UT control group (28.5 ± 2.5 cells/100 μm). The mean goblet cell densities were 15.1 ± 3.1 cells/100 μm in the BSS group (P = 0.59 compared with the EDE group), 16.2 ± 3.0 in the 0.0001% adiponectin group (P = 0.63 compared with the EDE control; P = 0.55 compared with the BSS group), 26.1 ± 4.0 cells/100 μm in the 0.001% adiponectin group (P < 0.05 compared with the EDE or BSS group), and 27.3 ± 4.2 cells/100 μm in the 0.01% adiponectin group (P < 0.05 compared with the EDE or BSS group) (Figs. 3A, 3B). 
Figure 3. 
 
Mean goblet cell densities (A) and representative figures (B) in the experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated groups. *P < 0.05, **P < 0.01 compared with the EDE group. †P < 0.05, ††P < 0.01 compared with the BSS group.
Figure 3. 
 
Mean goblet cell densities (A) and representative figures (B) in the experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated groups. *P < 0.05, **P < 0.01 compared with the EDE group. †P < 0.05, ††P < 0.01 compared with the BSS group.
Antigens of adiponectin receptors, AdipoR1 and AdipoR2, were distinctly immunodetected in the conjunctiva and lacrimal gland of normal eyes (Figs. 4A–D). 
Figure 4. 
 
Immunohistochemistry showing the expression of adiponectin receptors, AdipoR1 (A, C) and AdipoR2 (B, D), in the conjunctiva and lacrimal gland of normal untreated mice.
Figure 4. 
 
Immunohistochemistry showing the expression of adiponectin receptors, AdipoR1 (A, C) and AdipoR2 (B, D), in the conjunctiva and lacrimal gland of normal untreated mice.
In EDE mice, strong staining for TNF-α in the conjunctival and lacrimal gland epithelium was detected. Mice treated with 0.001% and 0.01% adiponectin solution showed weak TNF-α staining in the conjunctival and lacrimal gland compared with the other mouse groups (Figs. 5A, 5B). 
Figure 5. 
 
Immunohistochemistry for TNF-α in the conjunctiva (A) and lacrimal gland (B) of experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated mice.
Figure 5. 
 
Immunohistochemistry for TNF-α in the conjunctiva (A) and lacrimal gland (B) of experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated mice.
Flow Cytometric Analysis
The mean percentages of CD4+CXCR3+ T cells in the conjunctival and lacrimal gland significantly increased in the EDE group (59.40% ± 9.32% and 68.37 ± 10.12) compared with the UT group after 10 days of desiccating stress (28.43 ± 2.41% and 29.98 ± 2.01%; P < 0.05 and P < 0.05). 
The mean percentages of CD4+CXCR3+ T cells in the conjunctiva were 57.97% ± 11.37% in the BSS group (P = 0.68 compared with the EDE group), 57.37 ± 8.91% in the 0.0001% adiponectin group (P = 0.50 compared with the EDE control; P = 0.31 compared with the BSS group), 33.91% ± 6.91% in the 0.001% adiponectin group (P < 0.05 compared with the EDE or BSS group), and 30.11% ± 6.55% in the 0.01% adiponectin group (P < 0.05 compared with the EDE or BSS group) (Fig. 6A). 
Figure 6. 
 
Flow cytometry showing CD4+CXCR3+ T cells in the conjunctiva (A) and lacrimal gland (B) of experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated mice.
Figure 6. 
 
Flow cytometry showing CD4+CXCR3+ T cells in the conjunctiva (A) and lacrimal gland (B) of experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated mice.
The mean percentages of CD4+CXCR3+ T cells in the lacrimal gland were 63.14% ± 11.30% in the BSS group (P = 0.32 compared with the EDE group), 60.10 ± 9.23% in the 0.0001% adiponectin group (P = 0.47 compared with the EDE control; P = 0.29 compared with the BSS group), 34.23% ± 6.97% in the 0.001% adiponectin group (P < 0.05 compared with the EDE or BSS group), and 32.56% ± 6.21%, in the 0.01% adiponectin group (P < 0.05 compared with the EDE or BSS group) (Fig. 6B). 
Discussion
Adiponectin, also referred to Acrp30, AdipoQ, apM1, or GBP28, is composed of an amino-terminal collagen domain, a carboxy-terminal globular domain, and complement factor C1q-like globular domain. 34,35 The crystal structure of adiponectin carboxy-terminal globular domain reveals a striking resemblance to the structure of TNF-α. 36 This protein is secreted mainly from adipose tissue into the bloodstream and is abundant in plasma relative to many biological functions. 37  
Adiponectin exists as a full-length protein or a proteolytic cleavage product known as globular adiponectin. Full-length adiponectin can exist as three multimeric species: a trimer (low molecular weight), a hexamer (middle molecular weight), and a high molecular weight (HMW) form. 38,39 Three adiponectin receptors have been identified: AdipoR1, AdipoR2, and T-cadherin. 40,41 AdipoR1 has a high affinity for globular adiponectin, whereas AdipoR2 has an intermediate affinity for globular adiponectin. 42  
Besides its well-known antidiabetic, antiatherosclerotic, and anticancer properties, a growing body of evidence suggests that adiponectin also exerts anti-inflammatory effects. Adiponectin reduces the release of the proinflammatory cytokines IL-6, TNF-α, and IFN-γ from activated monocytes and macrophages and increases the secretion of anti-inflammatory mediators such as IL-10 and IL-1RA. 1820 Moreover, adiponectin can also inhibit the production of CXCR3 ligands in macrophages. 21 Although adiponectin is generally known to exhibit potent anti-inflammatory properties, it can also participate in the development of autoimmune RA by stimulating IL-6 and pro-matrix metalloproteinase 1 (proMMP1) production in synovial fibroblasts. 43 However, paradoxical elevation of serum adiponectin levels in systemic autoimmune diseases has been explained by a mechanism involved in the control of inflammatory or immunologic processes occurring in these disease states. 44 In addition, adiponectin can activate adenosine monophosphate-activated protein kinase (AMPK) and protect salivary gland epithelial cells from spontaneous and IFN-γ-induced apoptosis in autoimmune inflammation. 45  
Dry eye is an immune-mediated inflammatory disease, mediated primarily by CD4+ T cells. 46 CD4+ T cells infiltrate the ocular surface, where they secrete proinflammatory cytokines. These immunoinflammatory responses lead to further ocular surface damage and the development of a self-perpetuating inflammatory cycle. 47 Desiccating stress induces tear hyperosmolarity, activating intracellular signaling pathways that initiate the production of proinflammatory cytokines and chemokines, including IL-1β, IL-6, TNF-α, INF-γ, and CXCR 3 ligands. 6,32,48,49 Because inflammation plays a pivotal role in the pathogenesis of dry eye disease, the use of anti-inflammatory therapy, such as cyclosporine A and corticosteroids, has been gaining popularity. Topical cyclosporine A inhibits T cell activities such as release of inflammatory cytokines including IL-2 and IFN-γ, and reduces apoptosis markers and proinflammatory cytokines, thereby mitigating dry eye by improving clinical signs and decreasing inflammation in the ocular surface and lacrimal gland. 50 Corticosteroids can also improve dry eye symptoms and signs through their anti-inflammatory actions. 51 However, long-term use of corticosteroids is associated with the risk of ocular hypertension, cataract, delayed epithelial healing, and infectious keratitis. Desiccating stress has been shown to activate mitogen-activation protein kinase (MAPK) in corneal epithelial cells; this leads to a local release of proinflammatory mediators, resulting in disruption of corneal integrity and release of cytokines. 48,52,53 Adiponectin provokes the activation of adenosine monophosphate–activated protein kinase (AMPK) and the inhibition of various proinflammatory signaling pathways such as p38 MAPK. 54,55 Activation of AMPK can inhibit the nuclear factor–κB signaling, which plays key essential roles in regulating inflammation and immune responses. 56  
In the present study, topical application of globular adiponectin led to an improvement of clinical signs and an decrease of inflammation in the ocular surface and lacrimal gland of desiccating stress–induced dry eye. Firstly, we found that the adiponectin receptors, AdipoR1 and AdipoR2, were distinctly present in the conjunctival epithelia as well as ductal epithelia of lacrimal glands of normal mice. Tear production and conjunctival goblet cell density were higher, and corneal surface irregularity scores were lower, in 0.001% and 0.01% adiponectin-treated mice compared with EDE control and BSS-treated mice. IL-1β, IL-6, TNF-α, IFN-γ, and MIG concentrations, TNF-α expression, and percentages of CD4+CXCR3+ T cells both in the conjunctiva and in the lacrimal gland were significantly lower in 0.001% and 0.01% adiponectin-treated mice than control mice. In contrast, the 0.0001% adiponectin-treated group did not show significant differences in parameters when compared with the EDE or BSS group. We think that topical application of adiponectin improved the tear film and ocular surface parameters by inhibiting inflammatory cytokines, chemokines, and T cells in the conjunctiva and lacrimal gland. 
In dry eye pathology, the lacrimal functional unit, which is composed of the ocular surface epithelium and lacrimal gland, can become the target of the immune system and show signs of inflammation. 57 Several studies have shown that amounts of IL-1β and/or TNF-α increased in lacrimal gland tissues. 58 Theses cytokines can impair lacrimal gland secretion via direct inhibition of neural activity or neurotransmitter release. 59 We have showed herein that topical adiponectin eye drops led to significant inhibition of lacrimal gland inflammation, indicated by increased tear production and decreased expression of these cytokines as compared with the controls. 
Cytokines released by the infiltrating CD4+ T cells may lead to goblet cell loss in dry eye. The number of CD4+ T cells has an inverse correlation with the number of conjunctival goblet cells in desiccating stress. 27 It has been demonstrated that IL-6 and INF-γ can promote loss of goblet cells under desiccating stress. 60,61 Our data supported that topical adiponectin eye drops could reduce release of these cytokines, thereby preventing the loss of goblet cells in the conjunctiva. 
The therapeutic efficacy of topical adiponectin was supported further by decreased chemokines, chemokine receptors, and CD4+ T cells in the conjunctiva and lacrimal gland. We previously reported that expressions of Th-1 chemokine receptors, CXCR3 and CCR5, and their ligands were upregulated in the ocular surface of dry eye. 62 We also demonstrated that Th-1-associated CCR5+CD4+ cells and CXCR3+CD4+ cells, as well as Th-17-associated CCR6+CD4+ cells, increased in the conjunctiva of dry eye, whereas Th-2-associated CCR4+CD4+ cells did not increase. 6,63 In our study, we found that topical adiponectin could decrease CXCR3-associated ligand MIG expression and CXCR3+CD4+ cell numbers in the conjunctiva and lacrimal glands. 
In conclusion, the application of 0.001% and 0.01% adiponectin eye drops can improve tear production and ocular surface irregularities, decrease inflammatory cytokines on the ocular surface and lacrimal gland by suppressing inflammatory cytokine expression and infiltration of CXCR3+CD4+, and increase conjunctival goblet cell density in mouse experimental dry eye. Our results suggest that topical application of adiponectin may be useful for the treatment of dry eye disease. 
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Footnotes
4  These authors contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Footnotes
 Disclosure: Z. Li, None; J.M. Woo, None; S.W. Chung, None; M.-Y. Kwon, None; J.-S. Choi, None; H.-J. Oh, None; K.-C. Yoon, None
Figure 1. 
 
Mean tear volumes in the untreated (UT) control, experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated groups at days 5 and 10. *P < 0.05, **P < 0.01 compared with the EDE group. †P < 0.05, ††P < 0.01 compared with the BSS group.
Figure 1. 
 
Mean tear volumes in the untreated (UT) control, experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated groups at days 5 and 10. *P < 0.05, **P < 0.01 compared with the EDE group. †P < 0.05, ††P < 0.01 compared with the BSS group.
Figure 2. 
 
Mean corneal smoothness scores (A) and representative figures (B) in the untreated (UT) control, experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated groups at days 5 and 10. *P < 0.05, **P < 0.01 compared with the EDE group. †P < 0.05, ††P < 0.01 compared with the BSS group.
Figure 2. 
 
Mean corneal smoothness scores (A) and representative figures (B) in the untreated (UT) control, experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated groups at days 5 and 10. *P < 0.05, **P < 0.01 compared with the EDE group. †P < 0.05, ††P < 0.01 compared with the BSS group.
Figure 3. 
 
Mean goblet cell densities (A) and representative figures (B) in the experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated groups. *P < 0.05, **P < 0.01 compared with the EDE group. †P < 0.05, ††P < 0.01 compared with the BSS group.
Figure 3. 
 
Mean goblet cell densities (A) and representative figures (B) in the experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated groups. *P < 0.05, **P < 0.01 compared with the EDE group. †P < 0.05, ††P < 0.01 compared with the BSS group.
Figure 4. 
 
Immunohistochemistry showing the expression of adiponectin receptors, AdipoR1 (A, C) and AdipoR2 (B, D), in the conjunctiva and lacrimal gland of normal untreated mice.
Figure 4. 
 
Immunohistochemistry showing the expression of adiponectin receptors, AdipoR1 (A, C) and AdipoR2 (B, D), in the conjunctiva and lacrimal gland of normal untreated mice.
Figure 5. 
 
Immunohistochemistry for TNF-α in the conjunctiva (A) and lacrimal gland (B) of experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated mice.
Figure 5. 
 
Immunohistochemistry for TNF-α in the conjunctiva (A) and lacrimal gland (B) of experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated mice.
Figure 6. 
 
Flow cytometry showing CD4+CXCR3+ T cells in the conjunctiva (A) and lacrimal gland (B) of experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated mice.
Figure 6. 
 
Flow cytometry showing CD4+CXCR3+ T cells in the conjunctiva (A) and lacrimal gland (B) of experimental dry eye (EDE) control, balanced salt solution (BSS)-treated, 0.0001% adiponectin-treated, 0.001% adiponectin-treated, and 0.01% adiponectin-treated mice.
Table 1. 
 
Concentrations of IL-1β, IL-6, TNF-α, INF-γ, and MIG in the Conjunctiva of UT Control, EDE Control, BSS-Treated, 0.0001% Adiponectin-Treated, 0.001% Adiponectin-Treated, and 0.01% Adiponectin-Treated Groups
Table 1. 
 
Concentrations of IL-1β, IL-6, TNF-α, INF-γ, and MIG in the Conjunctiva of UT Control, EDE Control, BSS-Treated, 0.0001% Adiponectin-Treated, 0.001% Adiponectin-Treated, and 0.01% Adiponectin-Treated Groups
IL-1β, pg/mL IL-6, pg/mL TNF-α, pg/mL INF-γ, pg/mL MIG, μg/mL
UT 1.08 ± 0.40†§ 0.90 ± 0.19†‡ 0.50 ± 0.18*§ 25.88 ± 3.87†§ 1.99 ± 0.43†§
EDE 6.82 ± 1.01 2.95 ± 0.85 1.59 ± 0.39 47.93 ± 6.41 4.84 ± 0.88
BSS 7.29 ± 1.62 2.59 ± 0.39 1.38 ± 0.41 42.46 ± 5.91 4.77 ± 0.86
0.0001% adiponectin 6.81 ± 1.00 2.57 ± 0.43 1.21 ± 0.21 43.45 ± 3.03 4.25 ± 0.49
0.001% adiponectin 1.30 ± 0.33*‡ 1.02 ± 0.27*‡ 0.55 ± 0.24*‡ 31.95 ± 3.27*‡ 2.20 ± 0.50*‡
0.01% adiponectin 1.22 ± 0.31*‡ 0.99 ± 0.33*‡ 0.52 ± 0.30*‡ 31.44 ± 2.88*‡ 2.17 ± 0.46*‡
Table 2. 
 
Concentrations of IL-1β, IL-6, TNF-α, INF-γ, and MIG in the Lacrimal Gland of UT Control, EDE Control, BSS-Treated, 0.0001% Adiponectin-Treated, 0.001% Adiponectin-Treated, and 0.01% Adiponectin-Treated Groups
Table 2. 
 
Concentrations of IL-1β, IL-6, TNF-α, INF-γ, and MIG in the Lacrimal Gland of UT Control, EDE Control, BSS-Treated, 0.0001% Adiponectin-Treated, 0.001% Adiponectin-Treated, and 0.01% Adiponectin-Treated Groups
IL-1β, pg/mL IL-6, pg/mL TNF-α, pg/mL INF-γ, pg/mL MIG, μg/mL
UT 26.47 ± 3.98†§ 30.73 ± 5.21†§ 0.79 ± 0.26†§ 83.43 ± 11.31*‡ 40.98 ± 6.21†§
EDE 47.98 ± 5.12 70.37 ± 7.23 3.99 ± 0.72 151.21 ± 20.23 90.45 ± 10.06
BSS 45.31 ± 4.31 60.11 ± 9.21 3.51 ± 0.78 132.37 ± 19.08 75.32 ± 9.23
0.0001% adiponectin 40.53 ± 2.09 59.79 ± 4.61 3.61 ± 0.45 137.45 ± 10.33 70.11 ± 6.20
0.001% adiponectin 33.23 ± 4.23*‡ 36.13 ± 5.16*‡ 0.83 ± 0.20*‡ 94.62 ± 10.20*‡ 48.76 ± 7.13*‡
0.01% adiponectin 31.32 ± 4.50*‡ 34.42 ± 4.28*‡ 0.81 ± 0.22*‡ 89.57 ± 11.48*‡ 42.19 ± 6.23*‡
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