Experimental Eye Research 134 (2015) 53e62

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Caffeic acid phenethyl ester lessens disease symptoms in an experimental autoimmune uveoretinitis mouse model Jae-Hyeog Choi a, b, Kug-Hwan Roh a, b, Hana Oh b, c, Sol-Ji Park a, b, Sung-Min Ha a, b, Mi Seon Kang d, Ji-Hyun Lee b, So Young Jung e, Hyunkeun Song a, b, Jae Wook Yang b, c, **, SaeGwang Park a, b, * a

Department of Microbiology and Immunology, College of Medicine, Inje University, Busan, Republic of Korea Ocular Neovascular Disease Research Center, Busan Paik Hospital, Busan, Republic of Korea Department of Ophthalmology, Busan Paik Hospital, College of Medicine, Inje University, Busan, Republic of Korea d Department of Pathology, Busan Paik Hospital, College of Medicine, Inje University, Busan, Republic of Korea e Department of Dermatology, Haeundae Paik Hospital, College of Medicine, Inje University, Busan, Republic of Korea b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 October 2014 Received in revised form 12 February 2015 Accepted in revised form 16 March 2015 Available online 17 March 2015

Experimental autoimmune uveoretinitis (EAU) is an autoimmune disease that models human uveitis. Caffeic acid phenethyl ester (CAPE), a phenolic compound isolated from propolis, possesses antiinflammatory and immunomodulatory properties. CAPE demonstrates therapeutic potential in several animal disease models through its ability to inhibit NF-kB activity. To evaluate these therapeutic effects in EAU, we administered CAPE in a model of EAU that develops after immunization with interphotoreceptor retinal-binding protein (IRBP) in B10.RIII and C57BL/6 mice. Importantly, we found that CAPE lessened the severity of EAU symptoms in both mouse strains. Notably, treated mice exhibited a decrease in the ocular infiltration of immune cell populations into the retina; reduced TNF-a, IL-6, and IFN-g serum levels: and inhibited TNF-a mRNA expression in retinal tissues. Although CAPE failed to inhibit IRBPspecific T cell proliferation, it was sufficient to suppress cytokine, chemokine, and IRBP-specific antibody production. In addition, retinal tissues isolated from CAPE-treated EAU mice revealed a decrease in NF-kB p65 and phospho-IkBa. The data identify CAPE as a potential therapeutic agent for autoimmune uveitis that acts by inhibiting cellular infiltration into the retina, reducing the levels of pro-inflammatory cytokines, chemokine, and IRBP-specific antibody and blocking NF-kB pathway activation. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Experimental autoimmune uveoretinitis Caffeic acid phenethyl ester Interphotoreceptor retinal-binding protein Pro-inflammatory cytokine Nuclear factor-kappa B

1. Introduction Human uveitis is an ocular-inflammatory disease that can often lead to blindness. Experimental autoimmune uveoretinitis (EAU) is an animal model that shares many features with human uveitic disorders (Caspi et al., 1988; Nussenblatt, 1991). EAU is an organspecific, T cell-mediated autoimmune disease that can be induced by immunization with retinal antigens (Ags), including retinal SeAg and interphotoreceptor retinal-binding protein (IRBP), or by the adoptive transfer of retinal Ag-specific T lymphocytes (Caspi

* Corresponding author. Department of Microbiology and Immunology, College of Medicine, Inje University, Busan 614-735, Republic of Korea. ** Corresponding author. Department of Ophthalmology, College of Medicine, Inje University, Busan 614-735, Republic of Korea. E-mail addresses: [email protected] (J.W. Yang), [email protected] (S. Park). http://dx.doi.org/10.1016/j.exer.2015.03.014 0014-4835/© 2015 Elsevier Ltd. All rights reserved.

et al., 1986; Gregerson et al., 1986; Mochizuki et al., 1985). EAU is considered to be a predominantly Th1-mediated autoimmune disease. As such, it is thought that the inhibition of proinflammatory cytokines such as tumor necrosis factor-a (TNF-a) and interleukin-1b (IL-1b) could mitigate EAU symptoms (Caspi, 2002; Rizzo et al., 1996). Macrophages and neutrophils are known to be key mediators of tissue damage in several experimental autoimmune disease models, and in agreement, both macrophages and neutrophils are the major effectors of tissue damage in uveitis (Sonoda et al., 2003; Yamamoto et al., 2010). Nuclear factor-kappa B (NF-kB) is implicated in the pathogenesis of many autoimmune diseases, including rheumatoid arthritis, type I diabetes, and multiple sclerosis (Pai and Thomas, 2008). Indeed, it was reported that NF-kB attenuation could alleviate the symptoms of EAU following treatment with an NF-kB inhibitor (Kitamei et al., 2006).


J.-H. Choi et al. / Experimental Eye Research 134 (2015) 53e62

Polyphenolic compounds derived from natural products are well known to have many advantages, namely, anti-oxidant (Chan et al., 2000), anti-tumoral (Suganuma et al., 1999), and antiinflammatory properties (Martinez and Moreno, 2000). Caffeic acid phenethyl ester (CAPE, empirical formula C17H16O4, molecular weight 284.31 g mol1) is a phenolic compound isolated from the propolis of honeybee hives (Fig. 1) and has anti-viral, anti-tumoral, anti-inflammatory, and immunomodulatory properties (Gokalp et al., 2006; Ozer et al., 2005). Several investigators have demonstrated the therapeutic potential of CAPE in treating allergic asthma (Jung et al., 2008b) and experimental autoimmune encephalomyelitis (EAE)-induced oxidative stress (Ilhan et al., 2004) by inhibiting NF-kB activity. In addition, CAPE is a potent inhibitor of mitogen-induced T cell proliferation and lymphokine production (Ansorge et al., 2003), and can also induce leukocyte apoptosis to further modulate NF-kB activity (Orban et al., 2000). However, no reports have demonstrated the effects of CAPE treatment in a mouse model of EAU. In the current study, we demonstrated the therapeutic effects of CAPE in C57BL/6 and B10.RIII mice with fully developed EAU and provide mechanistic evidence for its suppressive effect on EAU pathogenesis. Notably, CAPE reduced immune cell infiltration into the retina and hindered IRBP-specific T cell cytokine, chemokine, and antibody production in a proliferation-independent manner. In addition, CAPE sufficiently inhibited NF-kB pathway activation. As such, we believe our study demonstrates the therapeutic potential of CAPE in EAU therapy. 2. Materials and methods 2.1. Mice and reagents B10.RIII mice (8- to 12-weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME). Wild-type C57BL/6 mice (8- to 10weeks old) were purchased from Orient Bio, Inc. (Sungnam, Korea). All mice were bred and maintained under specific pathogenfree conditions in the Animal Care Facility at the College of Medicine, Inje University. All experimental procedures were examined and approved by the Institutional Animal Care and Use Committee of Inje University (Protocol No. 2013-053). Human IRBP peptide spanning amino acid residues 161e180 (IRBP161e180, SGIPYIISYLHPGNTILHVD) and human IRBP peptide 1e20 (IRBP1e20, GPTHLFQPSLVLDMAKVLLD) were synthesized by Peptron (Daejeon, Korea). Complete Freund's adjuvant (CFA) containing 1.0 mg/mL Mycobacterium tuberculosis was purchased from SigmaeAldrich (St. Louis, MO). Purified Bordetella pertussis toxin (PTX) and CAPE were purchased from Sigma Chemical Co. (St. Louis, MO). CAPE was first dissolved in dimethylsulfoxide (DMSO) and then diluted in phosphate-buffered saline (PBS). 2.2. EAU induction and clinical evaluation B10.RIII and C57BL/6 mice were immunized subcutaneously (s.c.) in the thighs and tail base with human IRBP peptides emulsified in CFA in a total volume of 200 mL. Specifically, B10.RIII and

C57BL/6 mice were immunized with 50 mg of IRBP161180 or 250 mg of IRBP120, respectively. An additional intraperitoneal (i.p.) injection of 0.2 mg of PTX was administered in 200 mL of PBS to C57BL/6 mice on post-immunization (PI) days 0 and 2. Clinical evaluation of retinal inflammation was recorded every 2 or 3 days by funduscopic examination. The severity of EAU was graded from 0 to 4, as described previously (Caspi, 2003). The clinical scoring was based on vessel dilatation, number of white focal lesions in vessels, and extent of retinal vessel exudate, hemorrhage, and detachment. 2.3. CAPE treatment Mice were injected i.p. with 10 mg/kg CAPE or vehicle (Pramanik et al., 2013). CAPE treatments were started on PI day 7, when EAU was developed (mean clinical score, ~1). B10.RIII mice were treated daily for 14 days, whereas C57BL/6 mice were treated for 10 or 14 days. Vehicle groups were treated with an equivalent volume of PBS. 2.4. Retinal flat mounts Retinal flat mounts were prepared according to a described previously method (Li et al., 2011). Briefly, mice were anesthetized with an i.p. injection of ketamine (100 mg/kg) and xylazine (10 mg/ kg). The lateral canthus of the left orbit was chosen as the injection point. Gentle pressure was applied to the periorbital area with two fingers in order to expose the eye, and 100 mL of FITC-dextran (50 mg/mL in ultrapure water; SigmaeAldrich) was injected. Enucleated eyes were then fixed in 4% paraformaldehyde for 30 min at room temperature. The cornea, iris, lens, and vitreous were gently removed under a stereomicroscope (S6E; Leica, Wetzlar, Germany). Four radial incisions were made in the dissected retina, and it was flattened with a coverslip. 2.5. Histopathology Mouse eyes were collected on PI day 21, fixed in 10% buffered formalin solution (Sigma), and embedded in paraffin. Sections (68 mm) were prepared and stained with hematoxylin and eosin. The histological findings (original magnification,  100 and  200) were examined for inflammatory infiltration as well as retinal folding and detachment. 2.6. Flow cytometry For staining, cell samples were prepared from the retinas of EAU-induced mice on PI days 7, 14, and 21. Cells were incubated with the Fc blocker 2.4G2 for 5 min and stained with 0.5 mg conjugated antibodies as follows: CD45-APC (clone 104), and CD3-FITC (clone 145-2C11) for T cells; CD45-APC, CD11b-PE (clone M1/70), and F4/80-PE/Cy5 (clone BM8) for macrophages; and CD45-APC, CD11b-PE, and Ly6G-PE/Cy7 (clone 1A8) for neutrophils. FACS data were acquired on a FACS Canto II flow cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (TreeStar; Ashland, OR). All antibodies were obtained from eBiosciences, Inc. (San Diego, CA). 2.7. T cell proliferation assay

Fig. 1. Structure of caffeic acid phenethyl ester (2-cyclohexylethyl (E)-3-(3,4dihydroxyphenyl)prop-2-enoate).

For proliferation assays, cells isolated from the spleen and draining lymph nodes (DLN; the inguinal and iliac lymph nodes) were labeled with CFSE for 5 min at 37  C (Choi et al., 2006). Cells were washed twice with PBS, and then stimulated with IRBP peptide (1, 10, 20, or 50 mg/mL) or concanavalin A (Con A; 5 mg/mL,

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SigmaeAldrich). After 72 h, cells were stained with CD4-PE/Cy5, and the intensity was determined by flow cytometry. 2.8. Cytokine production Cytokine production was measured with the mouse Th1/Th2/ Th17 cytokine Cytometric Bead Array (CBA) kit (BD Biosciences) according to the manufacturer's instructions. 2.9. Reverse transcription-polymerase chain reaction The retinas were collected on PI day 9 and PI day 11 after IRBP immunizations. They were homogenized, and total RNA was purified using TRIzol reagent (Invitrogen Life Technologies; Carlsbad, CA) and quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific; Waltham, MA). cDNA was synthesized with 2 mg of RNA. The PCR reactions were performed using Accupower® premix (Bioneer; Daejeon, Korea). The specific primers used for PCR are as follows: for TNF-a, 50 -CCACACCGT CAGCCGATTTG-30 and 50 -CACCCATTCCCTTCACAGAGC-30 ; for CCR5, 50 -CGAAAACACATGGTCAAACG-30 and 50 -GTTCTCCT 0 0 GTGGATCGGGTA-3 ; for MIP-1b, 5 -GTTCTCAGCACCAATGGGCTC TGA-30 and 50 -CTCTCCTGAAGTGGCTCCTCCTG-30 ; for RANTES, 50 CATCCTCACTGCAGCCGCCC-30 and 50 -CCAAGCTGGGTAGGACTAGAG-30 ; and for GAPDH, 50 -TTCACCACCATGGAGAAGGC-30 and 50 GGCATGGACTGTGGTCATGA-30 . 2.10. Determination of IRBP-specific total IgG levels in sera Serum levels of IRBP-specific total IgG in immunized mice treated with or without CAPE were determined by ELISA, as previously described (Pennesi et al., 2003). Briefly, 96-well microtiter plates (Nunc, Denmark) were coated with IRBP (1 mg/mL); then, the plates were blocked with BSA and incubated with samples of the sera. Bound total IgG was detected by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and tetramethylbenzidine (TMB) substrate. Absorbance (450 nm) was measured with an ELISA plate reader, and the values have been represented in arbitrary OD units. 2.11. Western blot analysis The retinas were collected on PI day 21. Tissues were homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors (Thermo Scientific, Rockford. IL). Tissue homogenates (20 mg of protein) were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. The blot was then probed with 1 mg/ mL primary antibody against NF-kB p65, IkBa, phospho-IkBa, or GAPDH (Santa Cruz Biotechnology; Santa Cruz, CA). Horseradish peroxidase-conjugated anti-rabbit (Jackson Immuno Research Laboratories, Inc., West Grove, PA) or anti-mouse IgG (Santa Cruz Biotechnology) was used as a secondary antibody. 2.12. Statistical analysis Statistical analysis was performed using GraphPad Prism (Version 5 for Windows; GraphPad Software; San Diego, CA). P values were calculated using t-test. P < 0.05 was considered statistically significant. 3. Results 3.1. CAPE mitigates the severity of EAU in B10.RIII mice To determine whether CAPE treatment conferred therapeutic


effects on EAU, B10RIII mice that are highly susceptible to EAU were immunized with IRBP161-180 as previously reported (Hankey et al., 2001). EAU began to develop on approximately PI day 6 with the IRBP161180 peptide, and reached a plateau on PI days 15e40 (Fig. 2A). Fundoscopy images of EAU mice are shown in Fig. 2B. Notably, retinas exhibited some focal lesions on PI day 8. These lesions increased in size on PI day 15 and were accompanied by linear lesions, vasculitis, papilledema, and retinal detachments. Papilledema and retinal detachments were observed on PI day 33. CAPE (10 mg/kg) was administered starting on PI day 7 when the clinical scores were ~1, and continued for 14 days. The effects of the CAPE treatments were not noticeable until PI day 13. On PI day 15 (8 days of treatment), CAPE blocked the progression of EAU although retinal hemorrhages were still present (Fig. 2A, B). Clinical scores of CAPE-treated mice were 2.13 ± 0.48 on PI days 15 and 19; however, those of vehicle-treated mice were 3.42 ± 0.50 and 3.5 ± 0.45, respectively (Fig. 2A). Furthermore, the scores further decreased to 1.38 ± 0.48 on PI day 26 and 1.25 ± 0.50 on PI day 40, even after CAPE treatment was stopped. To assess the effects of CAPE on the retinal vasculature, flat-mounted preparations of FITC-Dextraninfused retinas harvested on PI day 40 were examined by microscopy. Marked areas of abnormal vasculature were detectable in the retinas from the vehicle group (Fig. 2C). In contrast, the retinas from CAPE-treated mice exhibited fewer abnormalities that were smaller than those in the vehicle group. In addition, retinal histology from PI day 21 showed massive inflammatory cell infiltrates in vehicletreated mice, yet only mild cellular infiltration was present in CAPE-treated mice (Fig. 2D). Furthermore, several retinal folds and detachments were observed in vehicle-treated mice; whereas the retinas isolated from CAPE-treated mice were nearly normal, with only a slight increase in the focal thickness of the retinal pigment epithelium layer.

3.2. CAPE lessens the severity of EAU in C57BL/6 mice To test whether the benefits of CAPE treatment could be observed in mice less susceptible mice to EAU, C57BL/6 mice were co-immunized with IRBP120 peptide and PTX. CAPE treatments were started on PI day 7, when the clinical scores were ~1, and continued for 10 days (Fig. 3A). EAU onset was observed at a similar time point in C57BL/6, compared to that of B10.RIII mice. However, overall disease progression was slower, with clinical scores peaking on PI day 22 (2.81 ± 0.26) through day 26 (2.81 ± 0.26), and decrease starting on PI day 30 (2.75 ± 0.27) in the vehicle group. Notably, EAU progression was further retarded in CAPE-treated mice (Fig. 3A). The scores showed significant differences from PI day 15 onward (day 15: 1.60 ± 0.32, day 18: 1.68 ± 0.26, day 20: 1.90 ± 0.32, day 22e26: 1.83 ± 0.41, and day 30: 1.75 ± 0.27; p < 0.05). Fundoscopy images of EAU-induced C57BL/6 mice are shown on PI day 30 in Fig. 3B. Focal or linear lesions, vasculitis, papilledema, and retinal detachment were observed in vehicletreated EAU C57BL/6 mice, whereas retinas appeared normal in naïve mice. However, focal, linear lesions and vasculitis only observed in CAPE treated mice (Fig. 3B). Retinal flat mounts of vehicle-treated mice displayed large areas of abnormal vasculature, whereas CAPE-treated mice only exhibited small numbers and sizes of these lesions (Fig. 3C). Retinal histopathology revealed an increase in retinal folds and inflammatory infiltrate in vehicle-treated mice, compared to the minor lesions and infiltrate observed in the CAPE-treated mice (Fig. 3D). In addition, no retinal or spotty lesions are observed in the fundus and histological examination from naïve C57BL/6 mice (Fig. 3B and D). These results indicate that CAPE is sufficient to suppress ocular inflammation and preserve retinal architecture in IRBP-induced EAU in C57BL/6 mice.


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Fig. 2. CAPE ameliorates the severity of EAU in B10.RIII mice. Mice were immunized subcutaneously (s.c.) in the thighs and tail base with 50 mg of IRBP (161e180) peptide in 0.2 mL of CFA emulsion containing 2.5 mg/mL Mycobacterium tuberculosis. CAPE (10 mg/kg, n ¼ 4) or vehicle (n ¼ 5) was administered intraperitoneally (i.p.) from PI days 8e20. (A) Clinical score of EAU. Comparison of the funduscopic EAU severity between CAPE- and vehicle-treated mice. Funduscopic examinations of mice were performed starting on day 1 after immunization. Focal lesions, linear lesions, vasculitis, retinal hemorrhages, papilledema and retinal detachment were examined. According to the severity of these findings, the EAU clinical scores were graded on a scale of 0e4. (B) Representative pictures of ocular fundus of the retina in CAPE- or vehicle-treated EAU mice are shown for days 8, 15, and 33. 1: focal lesions, 2: linear lesions, 3: vasculitis, 4: retinal hemorrhages, 5: papilledema and 6: retinal detachment (C) Images from the flat mount of FITC-Dextran-infused retinas from mice sacrificed on PI day 40. Numeral 1e6, or 1e3 is an enlarged photo from the left. (D) Histological sections through the retina on PI day 21 were stained with H&E. Retinal detachments were observed in retina (arrow). Data are mean ± SD. *, p < 0.05; **, p < 0.01; and ***, p < 0.001, compared with vehicle-treated controls. Results are representative of three independent experiments.

3.3. CAPE alters the retinal cell population in EAU mice To determine whether the immunosuppressive nature observed with CAPE treatment would be sufficient to alter the respective populations of ocular-infiltrating cells in EAU, retinal cell infiltrates were isolated by collagenase treatment and the percentages of leukocytes, T cells, macrophages, and neutrophils were analyzed by flow cytometry (Fig 4A). Ocular-infiltrating cells were harvested on PI days 7, 14, and 21. Since the proportions of CD45þ cells were very small, we analyzed pooled retinal cells from six mice and collected 1  106 events per pooled samples. CD45þ cells were

Caffeic acid phenethyl ester lessens disease symptoms in an experimental autoimmune uveoretinitis mouse model.

Experimental autoimmune uveoretinitis (EAU) is an autoimmune disease that models human uveitis. Caffeic acid phenethyl ester (CAPE), a phenolic compou...
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