Oral Administration of Retinoic Acid Receptor-α/β-Specific Ligand Am80 Suppresses Experimental Autoimmune Uveoretinitis

Abstract

Purpose.: To determine whether synthetic retinoic acid receptor (RAR)-α/β–specific agonist Am80 reduces inflammation in experimental autoimmune uveoretinitis (EAU).

Methods.: Naive CD4⁺ T cells were activated with anti-CD3, anti-CD28, and transforming growth factor (TGF)-β, in the presence or absence of Am80. Intracellular expression of forkhead box p3 (Foxp3) and interleukin (IL)-17 in the activated CD4⁺ T cells was assessed by flow cytometry. For induction of EAU, C57BL/6 mice were immunized with human interphotoreceptor retinoid binding protein (IRBP) peptide 1 to 20 (IRBP_1–20). Am80 was administered orally every other day (3 mg/kg/time point) from day 0 to day 21. In vivo primed draining lymph node cells from vehicle-treated or Am80-treated mice were stimulated with IRBP_1–20, and culture supernatant was harvested for assay of interferon (IFN)-γ, IL-6, IL-10, and IL-17. The expression of Foxp3 and IL-6 receptor α in CD4⁺ T cells of draining lymph node cells was assessed by a flow cytometer.

Results.: Am80 synergized with TGF-β to induce Foxp3⁺ T regulatory cells (Treg) and reciprocally inhibited development of IL-17–producing T helper cells (Th17) induced by TGF-β and IL-6. Am80 treatment reduced the severity of EAU clinically, and IFN-γ and IL-17 production was significantly reduced in Am80-treated mice. In addition, the expression of IL-6 receptor α on CD4⁺ T cells was downregulated in Am80-treated mice.

Conclusions.: These findings demonstrate that Am80 treatment ameliorates severity of EAU and reduces the Th1/Th17 responses. The synthetic retinoid Am80 appears to be a promising agent for preventing autoimmune uveoretinal inflammation.

Experimental autoimmune uveoretinitis (EAU) is an animal model that shares many clinical and histologic features with human uveitic disorders such as Behçet's disease and sarcoidosis. ¹ EAU is induced by immunization with a retinal antigen, such as S-antigen or interphotoreceptor retinoid-binding protein (IRBP) in complete Freund's adjuvant (CFA), or by adoptive transfer of retinal antigen-specific CD4⁺ T cells. ^2,3 Recent studies have shown that EAU can be driven by helper T (Th)1 cells or Th17 effector cells in mice. ^{4 –7} In addition, it has been reported that naturally occurring regulatory T cells (Tregs) have an immunoregulatory role in EAU. ^8,9  

Vitamin A (retinol) and its derivatives, referred to together as “retinoids,” are compounds that bind to and activate retinoid acid receptors (RARα, β, γ and RXRα, β, γ), members of the nuclear hormone receptor family. ¹⁰ Retinoids are specific modulators of cell proliferation, differentiation, and morphogenesis in vertebrates. ¹¹ Retinol is metabolized to retinal and then to all-trans retinoic acid (ATRA), an active metabolite of vitamin A, which binds to RARα, β, γ in the cell nucleus. Recently many functions of retinoids have been identified in regulation of immune responses. In vitamin A–deficient mice, ATRA shifts the immune response from a Th2-type to a Th1-type response. ¹² In addition, retinoids suppress Th1 development and enhance Th2 development in vitro. ^13,14 Several studies have demonstrated that retinoid treatment in vivo suppresses inflammation in autoimmune disease models. ^{15 –17} Recently ATRA has been shown to be a key regulator of transforming growth factor (TGF)-β–dependent immune responses, capable of inhibiting the interleukin (IL)-6–driven induction of Th17 cells and promoting a new subset of T cells expressing the transcription factor forkhead box p3 (Foxp3), called T regulatory cells (Tregs). ^{18 –20} These findings have focused attention on developing retinoids as possible drugs for immunomodulation and treatment of autoimmune disease. However, there are some problems with the clinical use of retinoids, such as acquired resistance to retinoic acid after continued administration and hypervitaminosis A with symptoms of diarrhea, vertigo, loss of hair, and skin desquamation. ²¹  

It is known that 4-[(5,6,7,8-tetrahydro-5,5,8,8,-tetramethyl-23-naphthyl) carbamoyl] benzoic acid (Am80) is a novel synthetic retinoic acid with potent binding activity to RAR-α and RAR-β but not RAR-γ. ²² Am80 has little affinity for cellular retinoic acid binding protein (CRABP) and is active against CRABP-rich ATRA-resistant cells. ²³ In addition, Am80 is more stable to light, heat, and oxidation than retinoic acid. It is reported that Am80 is effective in the treatment of relapsed acute promyelocytic leukemia. ²⁴  

The aims of the present study were to determine the effect of Am80 on Th17 and Treg differentiation in vitro and in vivo, and whether Am 80 treatment can affect the development of inflammation in EAU. Our results showed that Am80 suppressed the induction of Th17 cells and promoted the development of Tregs in vitro, and that oral administration of Am80 was effective in suppressing inflammation in EAU mainly by suppressing differentiation of Th1/Th17 effector cells. We also found that the expression of IL-6 receptor α on CD4⁺ T cells was reduced in Am80-treated mice. In addition, Am80 treatment even during effector phase was capable only of delaying inflammation in EAU. Furthermore, DNA microarray analysis demonstrated that Am80 treatment decreased the expression of inflammatory response–related genes in inflamed retina. These results suggest that Am80 may be a promising agent for preventing autoimmune uveoretinal inflammation mediated by the Th1/Th17 pathway. 

Materials and Methods

Mice and Reagents

Six-to-8-week-old female C57BL/6J mice were purchased from Japan CLEA (Shizuoka, Japan). All mice were treated in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. This study was approved by the institutional guidelines regarding animal experimentation. IRBP peptide 1–20 (GPTHLFQPSLVLDMAKVLLD) was obtained from Takara-Bio (Shiga, Japan). Am80 was provided by the Research Foundation ITSUU Laboratory (Tokyo, Japan). Am80 was suspended in 0.5% carboxymethyl cellulose solution for oral administration. All antibodies used for flow cytometry were obtained from BD Bioscience (San Jose, CA). All cytokines and TGF-β1 were purchased from R&D Systems (Minneapolis, MN). TGF-β1 was purified from human platelets, and the purity was >97% as determined by SDS-PAGE. 

Induction and Scoring of EAU

Mice were immunized subcutaneously in the neck region with 200 μg of IRBP_1–20 emulsified in 0.2 mL CFA (Difco, Detroit, MI) containing 1 mg Mycobacterium tuberculosis strain H37Ra (Difco), and given 100 ng pertussis toxin (Sigma-Aldrich, St. Louis, MO) intraperitoneally as an additional adjuvant. ²⁵ Am80 at a dose of 3 mg/kg/time point was administered orally every other day. Funduscopic examination was performed on days 13, 15, 17, 19, and 21 after immunization, and clinical findings were graded from 0 to 4 as described previously. ²⁶ Clinical score was assessed in a masked fashion. Eyes were enucleated on day 21, and inflammation was assessed histopathologically. 

Naive CD4⁺ T Cell Purification and Stimulation

Naive CD4⁺ T cells were purified by MACS beads isolation of CD4⁺ T cells (Miltenyi Biotec, Gladbach, Germany). The purity of CD4⁺ cells was >90% as assayed by flow cytometry. Naive CD4⁺ T cells (2 × 10⁵/well) were activated with anti-CD3 (10 μg/mL) and anti-CD28 (1 μg/mL) in 96-well plates for 72 hours. Am80 (1 μM) was included in the culture. Cultures were also supplemented with IL-2 (100 U/mL), IL-6 (20 ng/mL) plus TGF-β1 (20 ng/mL) for Th17 differentiation, and TGF- β1 (20 ng/mL) for Treg cell conversion. Naive CD4⁺ T cells (2 × 10⁵/well) were cultured for 72 hours for Th17 differentiation and Treg cell conversion. 

In Vitro Proliferation and Cytokine Assay

Cervical and axillary lymph node cells obtained from immunized mice on day 15 or 21 (2 × 10⁵ cells/well) were cultured in 0.2 mL RPMI 1640 (Sigma-Aldrich) containing 10 mM HEPES, 0.1 mM nonessential amino acid, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin (all from Invitrogen Life Technologies, Carlsbad, CA), 1 × 10^-5 M 2-mercaptoethanol (Sigma-Aldrich), 10% fetal bovine serum, and IRBP_1–20 (10 μg/mL). For the cytokine assay, supernatants were collected after 96 hours and analyzed for interferon (IFN)-γ, IL-6, and IL-10 using quantikine ELISA kits (R&D Systems) and for IL-17 using mouse IL-17 ELISA kits (Bender MedSystems, Vienna, Austria). Cell proliferation was evaluated using a cell proliferation assay (bromodeoxyuridine; Roche Diagnostics, Mannheim, Germany). 

Flow Cytometry

For intracellular cytokine staining, cells were stimulated in culture medium containing PMA (5 ng/mL; Sigma), ionomycin (500 ng/mL; Sigma), and monesin as cytokine secretion blocker (Gogi-stop; BD Bioscience, San Jose, CA) in a cell incubator with 10% CO₂ at 37°C for 4 hours, then stained using FITC-conjugated monoclonal antibodies against mouse CD4 (BD Bioscience). The cells were washed, fixed, permeabilized with buffer (Cytofix/Cytoperm; BD Bioscience), intracellularly stained with PE-conjugated antibodies against IL-17 (BD Bioscience), and analyzed on a flow cytometer (FACSCalibur; BD Bioscience) using acquisition and analysis software (CellQuest; Becton Dickinson, Franklin Lakes, NJ). For Foxp3 staining, cells were stained with FITC-conjugated antibodies against CD4. The cells were washed, fixed, permeabilized with buffer (eBioscience, San Diego, CA), intracellularly stained with PE-conjugated antibodies against Foxp3 (eBioscience), and analyzed on a flow cytometer. For staining of IL-6 receptor α (CD126) on CD4⁺ T cells, cells were stained with FITC-conjugated antibodies against CD4 (BD Bioscience) and PE-conjugated antibodies against IL-6Rα (CD126: BD Bioscience) and analyzed on a flow cytometer (FACSCalibur; BD Bioscience). 

DNA Microarray Hybridization and Analysis

Eyes were enucleated from vehicle-treated mice (3 mice) and Am80-treated mice (3 mice) on day 20 after immunization. Total RNA was extracted from retina of the enucleated 3 eyes in each group (Isogen RNA isolation kit; Nippon Gene, Tokyo, Japan) and purified (RNeasy Mini Kit; Qiagen, Tokyo, Japan). Total RNA was pooled within each group. Total RNA was analyzed (Agilent 2100 Bioanalyzer; Agilent Technologies, Santa Clara, CA), and a UV spectrophotometer was used to check the quality. Total RNA was taken 1 μg, and biotin-labeled cRNA was synthesized (MessageAmp II-Biotin Enhanced Kit; Ambion, Austin, TX). The biotin-labeled cRNA was fragmented and hybridized (CodeLink Mouse Whole Genome Bioarray; Applied Microarrays, Tempe, AZ) for 18 hours (300 rpm shaker) at 37°C. The hybridized slides were washed and incubated (streptavidin-Alexa Fluor 647; GE Health Care Bio-Science Corp., Piscataway, NJ) for 30 minutes at 25°C to label the cRNA and washed again. Then the slides were scanned (GenePix 4000B; Molecular Devices, Sunnyvale, CA). Scanned image files were analyzed using CodeLink Expression Analysis v5.0 software (Applied Microarrays). The net intensity was calculated by subtracting the median intensity of all pixels within the local background area from the mean intensity of all pixels within the spot areas. The net intensity of each spot was normalized by quantile using Microarray Data Analysis Tool v.3.2 software (Filgen, Nagoya, Japan), and gene expression data of retina from vehicle-treated or Am80-treated mice were compared. 

Statistical Analysis

Results of experiments were analyzed using the Mann-Whitney U test and Student's t-test. Means were considered to be significantly different for P < 0.05. 

Results

Promotion of Foxp3⁺ Treg Conversion and Inhibition of Th17 Differentiation by Am80

Recent studies have demonstrated that ATRA and synthetic retinoid promote the Foxp3⁺ Treg conversion and inhibit Th17 differentiation in vitro. ^18,19 To determine the effect of Am80 on Foxp3⁺ Treg cell and Th17 cell differentiation, we examined whether Am80 would affect the TGF-β induced development of Foxp3⁺ Treg and Th17 cells. As shown in Figure 1A, when purified CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 plus TGF-β, 40.6% of CD4⁺ T cells become Foxp3⁺ cells. Addition of Am80 increased the percentage of Foxp3⁺ T cells (56.8%). On the other hand, Am80 alone did not induce Foxp3 expression. Furthermore, under established Th17-favoring conditions (IL-6, TGF-β1), purified CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 plus IL-2, in the presence or absence of Am80. As shown in Figure 1B, in the absence of Am80, the conditions generated substantial proportions of IL-17–producing cells (10.3%), which were reduced by inclusion of this compound (2.6%). Thus, we confirmed that Am80 was able to enhance TGF-β induced Foxp3⁺ Treg conversion and inhibit IL-6– and TGF-β–induced Th17 differentiation. 

Figure 1.

 

Promotion of Foxp3 expression and inhibition of Th17 differentiation by Am80. Naive CD4⁺ T cells were activated with anti-CD3 and anti-CD28 for 3 days under indicated culture conditions in the presence or absence of Am80 (1 μM). Cultures were also supplemented with IL-2 (100 U/mL), IL-6 (20 ng/mL) plus TGF-β1 (20 ng/mL) for Th17 differentiation, and TGF- β1 (20 ng/mL) for Treg cell conversion. (A) Aliquots of cells were fixed, permeablized, and stained with PE-Foxp3 mAb and FITC-CD4 mAb and analyzed by flow cytometry. Numbers in upper right quadrants indicate the percentage of Foxp3-expressing CD4⁺ T cells in total CD4⁺ T cells. (B) Cells were reactivated with PMA and ionomycin for 4 hours in the presence of GolgiStop and stained with PE-IL-17 mAb and FITC-CD4 mAb and analyzed by flow cytometry. Numbers in upper right quadrants indicate the percentage of IL-17 producing CD4⁺ T cells in CD4⁺ T cells. This experiment was repeated twice with similar results.

Oral Administration of Am80 Reduces the Severity of EAU

Next we examined whether Am80 could suppress the development of EAU. Immunized mice were treated with vehicle or Am80 (3 mg/kg/mouse) every other day from day 0 to day 21 after immunization. The incidence and severity of EAU in vehicle- or Am80-treated mice were evaluated on days 15, 17, 19, and 21 after immunization. Fundus examination revealed that the severity of EAU was ameliorated in Am80-treated mice compared with vehicle-treated mice (Fig. 2A). Histopathological examination of eyes from vehicle-treated mice exhibited inflammatory changes in the posterior segments of EAU eyes. Inflammatory cell infiltration into the vitreous cavity and throughout all layers of the retina, retinal vasculitis, granuloma formation, and retinal folds were observed in vehicle-treated mice (Fig. 2B). In contrast, Am80-treated mice had significant reduction in the severity of EAU (Fig. 2B). Am80-treated mice showed significantly decreased severity of EAU by histopathological examination (Fig. 2C). These results clearly indicate that oral administration of Am80 is effective in suppressing inflammation in EAU. 

Figure 2.

 

Oral administration of Am80 during the entire phase reduces the severity of EAU. (A) Clinical score of EAU in vehicle- or Am80-treated mice. Immunized mice were treated with vehicle or Am80 from day 0 to day 21 after immunization. EAU findings were evaluated on days 15, 17, 19, and 21 after immunization (n = 8). The presented data indicates mean ± SE (SEM) within each group. Statistical analysis was performed by a Mann-Whitney U test. *P < 0.05 versus vehicle-treated mice. (B) Histopathological images of eyes enucleated from vehicle- or Am80-treated mice on day 21 after immunization. (C) Histopathological score of EAU in vehicle- or Am80-treated mice on day 21 after immunization. The presented data indicate mean ± SE (SEM) within each group. Statistical analysis was performed by a Mann-Whitney U test. *P < 0.05 versus vehicle-treated mice. This experiment was repeated three times with similar results.

Reduced Ag-Specific Th17 and Th1 Responses by In Vivo Treatment with Am80

To determine the mechanism of the suppressive effect of Am80, we examined Th1/Th17 responses in draining lymph node cells on day 15 after immunization. As shown in Figure 3A, although antigen-specific proliferation was not decreased in draining lymph node cells from Am80-treated mice, lymph node cultures from Am80-treated mice showed reduced production of IL-17 and IFN-γ compared with those from vehicle-treated mice (Fig. 3B). 

Figure 3.

 

Reduced Ag-specific Th17 and Th1 responses by Am80 treatment. (A) Antigen-specific proliferation of draining lymph nodes in vehicle- or Am80-treated mice. Immunized mice were treated with vehicle or Am80 from day 0 to day 15 after immunization. Draining lymph node cells collected on day 15 after immunization were pooled within each group (n = 4). Cultures were stimulated with IRBP_1–20 (10 μg/mL) for 96 hours and pulsed with bromodeoxyuridine for the last 24 hours. (B) Cytokine production of IL-17, IFN-γ, IL-6, and IL-10 by draining lymph node cells from vehicle- or Am80-treated mice. Immunized mice were treated with vehicle or Am80 from day 0 to day 15 after immunization. Draining lymph node cells collected on day 15 after immunization were pooled within each group (n = 4). Cultures were stimulated with IRBP_1–20 (10 μg/mL) for 96 hours, and supernatants collected at 96 hours were assayed by ELISA. The presented data indicate mean ± SE within each group. Statistical analysis was performed by Student's t-test. *P < 0.05 versus vehicle-treated mice. This experiment was repeated twice with similar results.

However, IL-6 and IL-10 levels in lymph node cultures were not significantly different between vehicle-treated mice and Am80-treated mice (Fig. 3B). These results suggest that administration of Am80 during the entire phase of EAU reduces both Th17 cells and Th1 immune responses. Furthermore, since we confirmed that Am80 enhanced TGF-β–induced Foxp3⁺ Treg conversion in vitro, we next examined the in vivo effect of Am80 on the Foxp3⁺ Treg cell population in these immunized mice. Immunized mice were treated with vehicle or Am80 from day 0 to day 21, and draining lymph nodes and spleens were collected on day 21 after immunization. Although Am80 treatment modestly increased the Treg population in cervical/axillary lymph nodes from immunized mice (Fig. 4), there was no significant difference with regard to the number of Tregs between vehicle-treated mice and Am80-treated mice (data not shown). Furthermore, Am80 treatment did not elevate the frequency of Foxp3⁺ Treg cells among CD4⁺ T cells in the spleen. These findings suggest that Am80 treatment may ameliorate uveoretinal inflammation by reducing the Th1/Th17 responses rather than induction of Tregs. 

Figure 4.

 

Am80 treatment in vivo increased the Foxp3⁺ Treg population in draining lymph node. Immunized mice were treated with vehicle or Am80 from day 0 to day 21 after immunization. Draining lymph nodes (cervical and axillary lymph nodes) and spleens were collected from each group (n = 4) on day 21 after immunization. The frequency of Foxp3⁺ CD4⁺ T cells was determined by flow cytometry. The presented data indicate mean ± SE within each group. *P < 0.05 versus vehicle-treated mice. NS, not significant. This experiment was repeated twice with similar results.

Reduced Expression of IL-6 Receptor α on CD4⁺ T Cells from Am80-Treated Mice

Recent work has shown that IL-6 is a target molecule for therapy in EAU. ²⁷ IL-6 receptors consist of the IL-6 receptor α (IL-6Rα) (CD126) and gp130 subunits. We examined whether oral administration of Am80 reduced the expression of IL-6Rα on CD4⁺ T cells of draining lymph nodes from immunized mice. Draining lymph nodes were collected from immunized mice treated with vehicle or Am80 on day 21 after immunization. As shown in Figure 5, the surface expression of IL-6Rα on CD4⁺ T cells was significantly reduced in Am80-treated mice. This result suggests that oral administration of Am80 during the entire EAU phase is capable of reducing expression of IL-6Rα on CD4⁺ T cells in draining lymph nodes from Am80-treated mice. 

Figure 5.

 

Reduced expression of IL-6 receptor α on CD4⁺ T cells from Am80-treated mice. Immunized mice were treated with vehicle or Am80 from day 0 to day 21 after immunization. Draining lymph nodes (cervical and axillary lymph nodes) were collected from each group (n = 4) on day 21 after immunization. The frequency of IL-6 receptor α (CD126)⁺ CD4⁺ T cells was determined by flow cytometry. The presented data indicate mean ± SE within each group. *P < 0.05 versus vehicle treated mice. This experiment was repeated twice with similar results.

Oral Administration of Am80 during the Effector Phase Delays the Progression of EAU

We next examined if Am80 was effective not only when administered throughout the entire period of EAU, but also after effector T cells had already been generated. Immunized mice were treated with vehicle or Am80 (3 mg/kg/mouse/time point) from day 8 through day 21 after immunization (the effector phase). EAU severity was evaluated on days 13, 15, 17, 19, and 21 after immunization. Fundus examination revealed that Am80 treatment significantly reduced the severity of EAU on days 15 and 17 after immunization (Fig. 6A). However, there was no significant difference with regard to the clinical score between vehicle-treated mice and Am80-treated mice on days 19 and 21 after immunization. Administration of Am80 during the effector phase also reduced IL-17 and IFN-γ production by primed lymph node cells, although inhibition of cytokine production did not attain statistical significance (Fig. 6B; IL-17, P = 0.15; IFN-γ, P = 0.06). There was no significant difference between both groups with regard to the proliferative response and IL-10 production by primed lymph node cells (Fig. 6B). We next tested the effect of Am80 on the expression of IL-6Rα on CD4⁺ T cells from primed lymph node cells on day 21. The expression of IL-6Rα on CD4⁺ T cells from primed lymph nodes was significantly reduced in Am80-treated mice (Fig. 6C). These results indicate that Am80 treatment during the effector phase alone is capable of delaying the progression of EAU, and that Am80 can suppress IL-17 and IFN-γ cytokine production and expression of IL-6Rα on CD4⁺ T cells from primed lymph nodes. 

Figure 6.

 

Oral administration of Am80 after effector cells have been generated (effector phase) delays the progression of uveoretinal inflammation in EAU. (A) Clinical score of EAU in vehicle- or Am80-treated mice. Immunized mice were treated with vehicle or Am80 from day 8 to day 21 after immunization. EAU findings were evaluated on days 13, 15, 17, 19, and 21 after immunization (n = 8). The presented data indicate mean ± SE within each group. Statistical analysis was performed by the Mann-Whitney U test. *P < 0.05 versus vehicle-treated mice. (B) Antigen-specific proliferation and cytokine production of draining lymph nodes in vehicle- or Am80-treated mice. Immunized mice were treated with vehicle or Am80 from day 8 to day 21 after immunization. Draining lymph node cells collected on day 21 after immunization were pooled within each group. Cultures were stimulated with IRBP_1–20 (10 μg/mL) for 96 hours and pulsed with bromodeoxyuridine for the last 24 hours. Supernatants collected at 96 hours were assayed by ELISA. The presented data indicate mean ± SE within each group. Statistical analysis was performed by Student's t-test. *P < 0.05 versus vehicle-treated mice. (C) Reduced expression of IL-6 receptor α on CD4⁺ T cells from Am80-treated mice during effector phase. Immunized mice were treated with vehicle or Am80 from day 8 to day 21 after immunization. Draining lymph nodes (cervical and axillary lymph nodes) were collected from each group (n = 4) on day 21 after immunization. The frequency of IL-6 receptor α (CD126)⁺ CD4⁺ T cells was determined by flow cytometry. The presented data indicate mean ± SE within each group. *P < 0.05 versus vehicle-treated mice. This experiment was repeated twice with similar results.

Reduced Expression of IL-6 Receptor α on CD4⁺ T Cells and Reduced IL-17 and IFN-γ Production in Draining Lymph Node Cells from Mice with EAU by Addition of Am80 into In Vitro Cultures

We further examined whether Am80 affected the expression of IL-6Rα on CD4⁺ T cells from immunized mice in vitro. Draining lymph nodes were collected and pooled from immunized mice. Lymph node cells were stimulated with human IRBP_1–20 for 96 hours. As shown in Figure 7A, the surface expression of IL-6Rα on CD4⁺ T cells was significantly decreased by addition of Am80. We next studied the effect of Am80 on antigen-specific Th17 and Th1 responses. Am80 suppressed IL-17 and IFN-γ production by draining lymph node cells stimulated with human IRBP_1–20 (Fig. 7B). Thus, Am80 treatment in vitro reduced the expression of IL-6Rα on CD4⁺ T cells and reduced IL-17 and IFN-γ production on antigenic restimulation. 

Figure 7.

 

Reduced expression of IL-6 receptor α on CD4⁺ T cells and reduced IL-17 and IFN-γ production in draining lymph node cells from mice with EAU by addition of Am80 into in vitro cultures. (A) Draining lymph node cells were collected from immunized mice (n = 4) on day 21 after immunization and pooled. Cultures were stimulated with IRBP_1–20 (Ag) (10 μg/mL) for 96 hours in the absence (Ag) or presence of Am80 (1 μM) (Ag+Am80). The frequency of CD126⁺ (IL-6 receptor α) CD4⁺ T cells in total CD4⁺ T cells was determined by flow cytometry. The presented data indicate mean ± SE within each group. Statistical analysis was performed by Student's t-test. *P < 0.05. (B) Draining lymph node cells collected on day 21 after immunization were stimulated with IRBP_1–20 (Ag) (10 μg/mL) for 96 hours in the absence (Ag) or presence of Am80 (1 μM) (Ag+Am80), and supernatants collected at 96 hours were assayed by ELISA (IL-17 and IFN-γ). The presented data indicate mean ± SE within each group. Statistical analysis was performed by Student's t-test. *P < 0.05. This experiment was repeated twice with similar results.

Comparison of Gene Expression in Inflamed Retina from Vehicle- and Am80-Treated Mice by DNA Microarray Analysis

To determine whether Am80 treatment alters gene expression of inflammatory cytokines, chemokines in inflamed retina, we compared RNA isolated from retina of vehicle- or Am80-treated mice. We used DNA microarrays that analyze 36,227 murine genes. The signal ratio (Am80-treated mice/vehicle-treated mice) of each of the 36,227 genes was calculated. Genes with a twofold increase in this ratio were defined arbitrarily as upregulated in retina from Am80-treated mice, whereas those with a twofold decrease in this ratio were defined as downregulated. Using these criteria, 648 genes were found to be upregulated, and 707 genes were found to be downregulated in retina from Am80-treated mice. Summaries of inflammatory response–related genes differentially expressed between two groups are shown in Table 1. Gene expression of IL-1β, IL-2 receptor gamma, IL-7, IL-7 receptor, IL-17 receptor D, IL-27, chemokine (C-C motif) ligand 5 (CCL5, RANTES: regulated on activation, normal T cell expressed and secreted), CCL6 (C10 protein), CCL8 (MCP-2: monocyte chemotactic protein-2), CCL19 (MIP-3β: macrophage inflammatory protein-3β), chemokine (C-X-C motif) ligand 9 (CXCL9, MIG: monokine induced by interferon gamma:), CXCL10 (IP-10: interferon γ-inducible protein 10), chemokine (C-X-C motif) receptor 4 (CXCR4), CD3 antigen, CD68 antigen, and Janus kinase 3 (Jak3) was downregulated in retina from Am80-treated mice. On the other hand, gene expression of nuclear factor kappa B (NF-κB) repressing factor (Nkrf) and mitochondrial superoxide dismutase 2 (SOD2) was upregulated in retina from Am80-treated mice. Thus, DNA microarray analysis demonstrated that Am80 treatment was capable of altering gene expressions of several inflammatory cytokines, chemokines, and transcriptional factors in inflamed retina. 

Table 1.

 

Representative Genes Differentially Expressed in Inflamed Retina from Am80-Treated Mice

Accession Number	Gene Symbol	Gene Name	Signal Ratio (versus Vehicle)
Downregulated genes
NM_009850	Cd3g	CD3 antigen, gamma polypeptide	0.06
NM_008372	Il7r	Interleukin 7 receptor	0.16
NM_021443	Ccl8	Chemokine (C-C motif) ligand 8 (MCP-2)	0.19
NM_013563	Il2rg	Interleukin 2 receptor, gamma chain	0.23
NM_010589	Jak3	Janus kinase 3	0.26
NM_013653	Ccl5	Chemokine (C-C motif) ligand 5 (RANTES)	0.26
NM_145636	Il27	Interleukin 27	0.29
NM_008361	Il1b	Interleukin 1 beta	0.30
NM_008371	Il7	Interleukin 7	0.34
NM_008599	Cxcl9	Chemokine (C-X-C motif) ligand 9 (MIG)	0.34
NM_021274	Cxcl10	Chemokine (C-X-C motif) ligand 10 (IP-10)	0.37
NM_009911	Cxcr4	Chemokine (C-X-C motif) receptor 4	0.38
NM_013487	Cd3d	CD3 antigen, delta polypeptide	0.39
NM_009139	Ccl6	Chemokine (C-C motif) ligand 6 (C10)	0.39
NM_009853	Cd68	CD68 antigen	0.39
AA537424	Ccl19	Chemokine (C-C motif) ligand 19 (MIP-3β)	0.42
NM_134437	Il17rd	Interleukin 17 receptor D	0.46
Upregulated genes
BY604766	Sod2	Superoxide dismutase 2, mitochondrial	3.29
AK020455	Nkrf	NF-kappaB repressing factor	3.36

Discussion

In the present study, we found that oral administration of Am80 was effective in suppressing inflammation in EAU, mainly by suppressing differentiation of Th1/Th17 effector cells. The expression of IL-6 Rα on CD4⁺ T cells was reduced in Am80-treated mice. In addition, Am80 treatment during the effector phase only was capable of delaying inflammation in EAU. Furthermore, DNA microarray analysis showed that Am80 treatment reduced the gene expression of inflammatory cytokines and chemokines in inflamed retina. 

Recently we have demonstrated that treatment using retinoic acid ameliorates EAU during the entire phase of the disease. ²⁸ Retinoic acid is a ligand for all RARs, the subtypes of which are designated RARα, RARβ, and RARγ. Am80 is a RARα/β-selective retinoid that does not bind and activate RAR-γ and RXRs, and hence can spare unfavorable adverse effects. Taken together, our data showed that Am80 treatment suppressed ocular inflammation, suggesting that RARα/β may be a promising target for therapeutics in autoimmune uveoretinal inflammation. Am80, clinically available in Japan for the treatment of acute promyelocytic leukemia, ²⁴ is superior to retinoic acid with regard to its chemical stability, agonist potency, lower affinity for cellular retinoic acid-binding proteins, and fewer potential side effects. In addition, Am80 can be administered orally, as shown in the present study. Thus, Am80 has additional therapeutic advantages compared with retinoic acid. 

The present study demonstrated that Th1/Th17 responses were reduced in Am80-treated mice and IL-6Rα expression on CD4⁺ T cells was decreased by Am80 treatment in vivo and in vitro, although oral administration of Am80 did not affect the production of IL-6 in draining lymph node cells stimulated with IRBP peptide. Since IL-6 is essential for differentiation of Th17 from naive T cell, ²⁹ reduced IL-6Rα expression on CD4⁺ T cells might be involved in reduced Th17 response in Am80-treated mice. Recently it has been shown that blockade of IL-6 ameliorate the severity of EAU by suppressing Th17 responses, suggesting that IL-6 is a therapeutic target for EAU. ²⁷ Taken together, it is conceivable that Am80 treatment may ameliorate uveoretinal inflammation by suppressing IL-6-driven signaling and Th17 response. 

We have recently shown that retinoic acid not only suppresses the Th1/Th17 response but also suppress antigen-specific proliferative responses, suggesting the possibility that activation of RARs by retinoids may induce systemic immune suppression. ²⁸ In contrast, our current data demonstrated that Am80 treatment during both the entire phase as well as the effector phase alone did not affect proliferative responses in draining lymph node cells. This is consistent with a recent report showing that cellular proliferation was not altered between Am80- or vehicle-treated mice. ³⁰ Taken together, it is likely that Am80 has the ability to regulate immune response by affecting antigen-specific Th1/Th17 responses in vivo without acting as a systemic immunosuppressive agent. 

We found that oral administration of Am80 during the effector phase only was capable of delaying the progression of EAU, but that Am80 treatment did not alter the severity of EAU on day 21. This is consistent with a recent report showing that Am80 treatment was ineffective at inhibiting late findings in experimental autoimmune encephalomyelitis (EAE). ³⁰ In addition, this report also showed that IL-10 production by central nervous system–infiltrating T cells was reduced in Am80-treated mice. ³⁰ Our study demonstrated that although IL-17/IFN-γ production by draining lymph node cells was suppressed in Am80-treated mice, IL-10 production was not altered. We hypothesize that Am80 treatment after effector cells have been generated may be suppressing the antigen-specific Th1/Th17 responses in peripheral lymphoid tissues, but it may not be able to prevent infiltration of antigen-specific and nonspecific effector cells into ocular lesions, resulting in damage to the blood-retinal barrier and progression of EAU. Our current data also showed that administration of Am80 during the entire phase ameliorated the clinical severity of EAU, indicating the possibility that continuous treatment by Am80 has the ability to suppress induction or priming of pathogenic effector T cells in EAU. 

The present study showed that although Am80 significantly increased the frequency of Foxp3⁺ Tregs in the presence of TGF-β, Am80 alone did not induce Foxp3 expression in vitro experiment. In addition, we have found that there is no induction of Tregs in spleen and quite modest elevation of Tregs in draining lymph nodes from Am80-treated mice. These results are compatible with previous reports showing a small increase in Tregs by activation of retinoic acid receptor-α/β, and this might be due to a lack of TGF-β in peripheral lymphoid organs in vivo. ^17,19 Using this speculation, it is most likely that Am80 treatment may ameliorate uveoretinal inflammation by reducing the Th1/Th17 responses rather than induction of Tregs. 

To determine whether Am80 treatment alters local immune response in inflamed retina, we compared RNA isolated from retina of vehicle- or Am80-treated mice using DNA microarrary analysis. The microarrray screening revealed reduced gene expression in several inflammatory cytokines (IL-1, IL-2 receptor, IL-7, IL-7 receptor, IL-17 receptor D, IL-27), chemokines (RANTES, C10, MCP-2, IP-10, MIG), and surface molecules for inflammatory cells (CD3: T cell, CD68: monocyte/macrophage). These findings are in agreement with the histopathological observations that Am80-treated mice exhibited only a few infiltrating cells in the vitreous cavity with the retinal layers remaining intact. Previously we reported that IP-10 and RANTES gene expressions were elevated in posterior segment of eyes with EAU. ³¹ Since the receptor of IP-10 and MIG, CXCR3, is predominantly expressed on memory or activated T cells, especially Th1 cells, ³² decreased expression of Th1-associated chemokines (IP-10 and MIG) in inflamed retina of Am80-treated mice may be associated with the reduced inflammatory cell infiltration in the posterior segment of eyes with EAU. Interestingly, the antioxidant gene, mitochondrial SOD2, was increased by approximately threefold in Am80-treated mice retina compared with vehicle-treated mice. This gene is a good candidate for future study in EAU mice because it is reported that increasing mitochondrial SOD expression provides long-term neuroprotection against EAE in the optic nerve, ³³ and upregulation of the mitochondrial SOD2 gene in retina of Am80-treated mice may play a potential role in retinal protection of the eyes with ocular inflammation, as well as in immune regulation. 

In conclusion, oral administration of Am80, a RAR-α/β–specific agonist, reduced uveoretinal inflammation in EAU mainly by inhibiting antigen-specific Th1/Th17 immune responses without affecting proliferative responses. In addition, Am80 treatment was capable of reducing the expression of IL-6 Rα on CD4⁺ T cells in vivo. Recent reports have demonstrated that Am80 has the potential value for treatment of animal models of autoimmune diseases. ^30,34 Our data suggest that activation of RAR-α/β may be a molecular target for the treatment of uveoretinal inflammation induced by Th1- or Th17-dominated inflammatory responses. 

Footnotes

 Supported by Grant-in-Aid No. 17791258 for Scientific Research from the Japan Society for the Promotion of Science. The Research Foundation ITSUU Laboratory provided the Am80 for this study.

Footnotes

 Disclosure: H. Keino, None; T. Watanabe, None; Y. Sato, None; A.A. Okada, None