Treatment of experimental autoimmune uveoretinitis with peroxisome proliferator-activated receptor α agonist fenofibrate.

PURPOSE: The peroxisome proliferator-activated receptor α (PPARα) agonist has been approved for treating hypercholesterolemia and lipid abnormalities. Researchers have recently discovered that an anti-inflammatory effect of PPAR agonist may have the potential to treat autoimmune disease. This study aims to investigate the therapeutic effects of fenofibrate on experimental autoimmune uveoretinitis (EAU).
METHODS: EAU was induced in Lewis rats using bovine S-antigen (S-Ag) peptide. Fenofibrate was suspended in 3% arabic gum and administered orally at a high dose of 100 mg/kg and at a low dose of 20 mg/kg every day. Fenofibrate treatment was initiated after the clinical onset once daily for 14 days. The rats were examined every other day for clinical signs of EAU. The histological scores and delayed-type hypersensitivity (DTH) were evaluated on day 28 post-immunization. Morphologic and immunohistochemical examinations were performed with light and confocal microscopy, respectively. Lymphocyte proliferation was measured with [3H] thymidine incorporation into antigen-stimulated T cells from inguinal lymph nodes.
RESULTS: Clinical and histological scores of EAU were decreased in the fenofibrate-treated groups. The expression of inflammatory cytokines and Müller cell proliferation were inhibited in the fenofibrate-treated groups. DTH was significantly inhibited in the fenofibrate-treated groups, compared with the vehicle-treated groups (controls). Lymphocyte proliferation assay demonstrated decreased proliferation in the presence of 25 mg/ml S-Ag peptide in the fenofibrate-treated groups compared with controls.
CONCLUSIONS: The current results indicate that fenofibrate administered orally following clinical onset has therapeutic effect in EAU. Fenofibrate may be useful for treating intraocular inflammation.

Introduction

Uveitis is a common cause of human visual disability and blindness. Experimental autoimmune uveoretinitis (EAU) is an organ-specific autoimmune disease that serves as an animal model of human uveitis and can be induced by immunization with retinal antigen (Ag; S-Ag or interphotoreceptor retinoid-binding protein [IRBP]) emulsified with complete Freund’s adjuvant (CFA) [1]. EAU is a CD4+ T cell-mediated disease in which macrophages play an important role as effector cells that generate intraocular inflammation and photoreceptor cell damage [1-3]. Breakdown of the blood–retina barrier leads to inflammatory cell infiltration of the anterior and posterior segments of the eye, followed by irreversible destruction of retinal photoreceptors [4].

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily, which includes steroid, retinoic acid, and thyroid hormone receptors [5]. Three isoforms of PPARs have been identified: PPARα, PPARγ, and PPARβ/δ. PPARα, the first PPAR to be cloned, regulates lipid homeostasis and is a target of the class of drugs known as fibrates [6-9]. Fibrates, such as fenofibrate and gemfibrozil, are used clinically for treating hyperglycemia and are safe and well tolerated by patients. PPARα is expressed in immune cells, including macrophages, dendritic cells, and T and B lymphocytes, and PPARα agonists are thought to play a role in regulating the inflammatory response [10-14]. PPARα agonists may also regulate inflammation by sustaining expression of the negative regulator nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα), thus preventing nuclear translocation and activation of nuclear factor kappaB (NF-κB), a major transcription factor involved in initiating proinflammatory immune responses [10,15]. Lovett-Racke et al. have shown that oral administration of PPARα agonists prevents the development of experimental autoimmune encephalomyelitis (EAE) [16].

The aim of the present study was to investigate the efficacy of treatment with fenofibrate in Lewis rats with EAU. This drug was administered after the first signs of disease development, and the effect was evaluated with clinical and histological scores, immunohistochemistry, lymph node cellular proliferation, and the development of delayed-type hypersensitivity (DTH).

Methods

Animals and anesthesia

Male Lewis rats weighing 200–250 g (Sankyo Labo Service, Tokyo, Japan) were used in the study. All experiments were conducted in accordance with the Animal Care Committee of the Jikei University School of Medicine and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Rats were anesthetized with a mixture (1:1) of ketamine hydrochloride (10 mg/kg; Wako Pure Chemicals Industries, Osaka, Japan) and xylazine hydrochloride (4 mg/kg; Wako).

Induction and evaluation of experimental autoimmune uveoretinitis

Each rat was immunized with a subcutaneous injection of 50 μg of bovine S-antigen (S-Ag) peptide 303–320 (DTNLASSTIIKEGIDKTV; purity >95%; Takara-Bio, Ohtsu, Japan), which has been shown to induce EAU [17], emulsified in the same volume of CFA (2 mg/ml) containing Mycobacterium tuberculosis H37Ra (Difco Laboratories, Detroit, MI). The rats were examined every other day for clinical signs of EAU and were graded 0–4 for clinical severity, as described previously [18]: grade 0, no inflammation; grade 1, iris vessel engorgement and minimal retinal vasculitis; grade 2, anterior chamber cells and mild retinal vasculitis; grade 3, fibrous exudates at the pupil margin and moderate retinal vasculitis; and grade 4, retroiridal hypopyon and severe retinal vasculitis. Topical 1% tropicamide and 2.5% phenylephrine hydrochloride were instilled to induce mydriasis to observe the fundus. Ocular examination was conducted using slit-lamp biomicroscopy and an indirect ophthalmoscope. The presence of inflammatory cells or fibrin in the anterior chamber, vessel dilatation, vessel tortuosity, or retinal hemorrhage in the fundus was scored in a blinded fashion by two ophthalmologists (M.O. and T.S.).

Treatment of experimental autoimmune uveoretinitis

Fenofibrate (Lipidil, Daiichi Sankyo, prescription formulation) was dissolved immediately before use in 3% arabic gum (Wako) and administered by oral gavage every day at a high dose of 100 mg/kg per rat and at a low dose of 20 mg/kg per rat. Alternatively, vehicle alone was administered orally daily. The EAU rats treated with vehicle alone were used as positive controls for comparison with the fenofibrate-treated groups. Fenofibrate treatment was initiated on day 14 post-immunization, when the first signs of clinical onset are usually observed. Daily administration was continued until day 27. On day 28, the animals were euthanized following assessment of DTH.

Histopathology and immunohistochemistry

The histopathology and immunohistochemistry methods have been described elsewhere [18,19]. Rats were euthanized with sodium pentobarbital (i.v.; Wako) on day 28 post-immunization, and the enucleated eyes were immersion fixed for 10 min in 4% paraformaldehyde in sodium cacodylate buffer (0.1 N; pH 7.4; Wako). After the cornea and lens were removed, the eyecup was cut into halves. One half of the tissue was stored in the fixative solution, and small areas of the retina were excised and embedded in low-melting point agarose (Sigma-Aldrich, St. Louis, MO) for immunocytochemical analysis with confocal microscopy. The embedded sections were cut on a Vibratome (VT1000S, Leica Microsystems, Heerbrugg, Switzerland) and blocked overnight in normal donkey serum (Jackson Immunoresearch Laboratories, West Grove, PA; 1:20) at 4 °C. The sections were then incubated overnight at 4 °C on a rotator with the following primary antibodies: mouse monoclonal antibodies (mAb) to interleukin (IL)-6 (R&D Systems, Minneapolis, MN; 1:100), IL-17 (R&D Systems; 1:100), and glutamine synthetase (GS; Chemicon, Temecula, CA; 1:100), and a rabbit polyclonal Ab to vascular endothelial growth factor (VEGF; Santa Cruz Biotechnology, Santa Cruz, CA; 1:100). All antibody solutions were prepared in PBTA (0.1 M PBS (1X; 140 mM NaCl, 2.7 mM KCl, 10 mM PO43-, pH 7.4) containing 0.5% bovine serum albumin [BSA; Fisher Scientific, Pittsburgh, PA], 0.1% Triton X-100 [Boehringer-Mannheim, Indianapolis, IN[, and 0.1% sodium azide [Sigma]). Control sections were rinsed in PBTA and then incubated with mouse and rabbit immunoglobulin G (IgG; Sigma). All antibody solutions were made in PBTA containing 0.5% BSA, 0.1% Triton X-100, and 0.1% sodium azide. After rinsing with PBTA, the sections were incubated with Cy3-conjugated donkey anti-mouse IgG (IL-17, GS) or Cy2-conjugated donkey anti-rabbit IgG (IL-6, VEGF; Jackson Immunoresearch Laboratories) overnight at 4 °C on a rotator. The sections were mounted in mounting medium for fluorescence (Vectashield, Vector Laboratories, Burlingame, CA) and viewed on a laser scanning confocal microscope (Laser Scanning System LSM510; Carl Zeiss Meditech, Oberkochen, Germany). For immunohistochemistry, both eyes were evaluated, and five histological sections from each animal (n=4) were examined.

For high-resolution transmission light microscopy analysis, the other half of the eyecup was immersion fixed in 1% glutaraldehyde (Wako) and 1% paraformaldehyde (Wako) in sodium phosphate buffer (0.086 M; pH 7.3) overnight at 4 °C, then fixed in phosphate-buffered osmium tetroxide (2%; Nisshin-EM, Tokyo, Japan) for 1 h, and embedded in epoxy resin (Nisshin-EM). These specimens were sectioned at 1 μm and stained with toluidine blue (Wako). Both eyes were evaluated. Five histological sections from each animal (n=5) were examined, and the histological severity of EAU was graded semiquantitatively in a blinded fashion by two ophthalmologists (M.O. and T.S.) on a scale of 0–7, as described previously [18,20]: grade 0, no tissue destruction; grades 1–2, destruction of outer segments of rods and cones; grades 3–4, destruction of the outer nuclear layer; grades 5–6 destruction of the inner nuclear layer; and grade 7, destruction of the ganglion cell layer.

Delayed-type hypersensitivity

The DTH response against the S-Ag peptide has been described elsewhere [18,21]. On day 26 post-immunization, the rats received an injection of 200 μg/20 μl of S-Ag peptide suspended in PBS into the right ear pinnae. After 48 h, ear swelling was measured using a micrometer (Mitsutoyo, Tokyo, Japan).

Lymphocyte proliferation assay

Lymph node cells (LNCs; 6×106 cells/ml) were prepared from control- (vehicle) or fenofibrate-treated rats on day 28 post-immunization, and grown in RPMI-1640 medium (Immuno-Biology Laboratories, Tokyo, Japan). Lymph node cells were pooled within the groups from vehicle- and fenofibrate-treated rats (n=4, each). Duplicate cultures were stimulated in vitro with S-Ag peptide. Cells were unstimulated or stimulated with 1.0, 5.0, and 25.0 μg/ml S-Ag peptide for 72 h. For the final 18 h, 1 mCi of [methyl-3H] thymidine (ICN, Costa Mesa, CA) was added to each well. The cells were harvested, and [3H] thymidine uptake was determined by measurement of the radioactivity with a Beta-plate reader (Beckman Instruments, Fullerton, CA).

Statistical analysis

We compared the EAU scores, DTH, and lymph node cellular proliferation using the non-parametric Mann–Whitney U-test. All results are expressed as mean ± standard deviation (SD) in the text and figures. P values less than 0.05 were considered statistically significant.

Results

Clinical and histological assessment

Following induction of EAU, animals were monitored every other day for clinical signs of disease. Clinical scores of day 17 and day 21 post-immunization are shown (Figure 1A,B). The high dose of fenofibrate (100 mg/kg) significantly decreased the clinical score compared with the vehicle-treated group, but the low-dose fenofibrate (20 mg/kg) did not significantly decrease the clinical score on day 17 or 21 post-immunization (Figure 1A,B). The fenofibrate-treated rats exhibited low-grade histological severity (Figure 2A–D). The mean EAU histological severity grade of the high-dose fenofibrate-treated rats was significantly low compared with the control rats and the low-dose fenofibrate-treated rats (Figure 2D). Control rats exhibited severe posterior uveitis with infiltration of inflammatory cells in the retina and destruction of the photoreceptor cell layer (Figure 2A). Rats treated with high-dose fenofibrate (100 mg/kg) exhibited suppression of inflammation and preservation of the photoreceptor cell layer, the target of the immune response. Meanwhile, photoreceptor destruction was not prevented completely by oral fenofibrate at 20 mg/kg or 100 mg/kg, since the treatment was started when the first sign of clinical onset was seen.

Figure 1

Clinical scores of rats with experimental autoimmune uveoretinitis. Clinical scores for days 17 (A) and 21 (B) post-immunization are shown. The clinical scores of the rats treated with high-dose fenofibrate (FF(100); 100 mg/kg, n=12)) were significantly lower than those of the rats treated with vehicle (3% arabic gum, n=12) or low-dose fenofibrate (FF (20); 20 mg/kg, n=12) on days 17 and 21 post-immunization. *, p=0.0001, **, p=0.0007, ***, p=0.0015.

Figure 2

Histopathologic features of the retina and histological scores of rats with experimental autoimmune uveoretinitis. The average histological score of five histological sections from each animal (n=5) was examined. Representative photographs of the groups treated with vehicle (A), low-dose fenofibrate (FF (20); 20 mg/kg) (B), and high-dose fenofibrate (FF (100); 100 mg/kg) (C) on day 28 post-immunization are shown. The vehicle-treated rats had severe posterior uveoretinitis with destruction of the photoreceptor cell layer with inflammatory cell infiltration in the retina. Rats treated with low-dose fenofibrate exhibited mild inflammatory cell infiltration. Rats treated with high-dose fenofibrate exhibited suppression of inflammation and preservation of the photoreceptor layer. INL; inner nuclear layer, ONL; outer nuclear layer (original magnification, ×100). The mean experimental autoimmune uveoretinitis (EAU) histological severity grades of the rats treated with vehicle, low-dose fenofibrate, and high-dose fenofibrate were 4.32±1.65 (n=5), 4.32±1.65 (n=5), and 4.32±1.65 (n=5), respectively. The scores of the rats treated with high-dose fenofibrate (FF(100)) were significantly lower than that of vehicle- or low-dose fenofibrate-treated rats (D). The results are presented as the mean ± standard deviation (SD) * p<0.0001 versus vehicle-treated group, ** p<0.0001 versus the low-dose fenofibrate-treated group.

Immunohistochemistry

Representative images of immunohistochemical staining of the retinas of the rats with EAU on day 28 post-immunization are shown in Figure 3. In rats with EAU treated with vehicle alone, expression of IL-6 (green) and IL-17 (red) was found in all layers of the retina (Figure 3A). Most of the infiltrated cells expressed IL-17. Treatment with low-dose fenofibrate (20 mg/kg) slightly reduced the number of infiltrated cells and the level of cytokines in the outer retina (Figure 3B). In rats with EAU treated with high-dose fenofibrate (100 mg/kg), expression of IL-17 and infiltration of cells were largely decreased in the retina (Figure 3C). In Figure 3A–C, there was some light immunoreactivity with the IL-17 antibody in the retinal pigment epithelium and IL-6 antibody in the inner limiting membrane, but this was the same in all preparations.

Figure 3

Immunohistochemistry of retinal sections from rats with experimental autoimmune uveoretinitis treated with vehicle (n=4), low-dose fenofibrate (n=4), and high-dose fenofibrate (n=4) on day 28 post-immunization. Marked expression of interleukin (IL)-6 (green) and IL-17 (red) was observed in ocular infiltrative cells in rats treated with vehicle (A). Rats treated with a high dose of fenofibrate showed weak expression of IL-6 and IL-17, and marked decrease in ocular infiltrative cells. INL; inner nuclear layer, ONL; outer nuclear layer, RPE; retinal pigment epithelium. The bar represents 50 µm.

Immunohistochemical results for retinas collected on day 28 post-immunization from rats treated with vehicle and fenofibrate are shown in Figure 4. In the vehicle- or low-dose fenofibrate-treated retinas, GS (red) and VEGF (green) were expressed in activated Müller cells (Figure 4A,B). Rats treated with high-dose fenofibrate (100 mg/kg) showed less VEGF expression (Figure 4C).

Figure 4

Immunohistochemistry of the retina of rats with experimental autoimmune uveoretinitis. Representative results for expression of vascular endothelial growth factor (VEGF; green) and glutamine synthetase (GS; red) are shown on day 28 post-immunization treated with vehicle (A), low-dose fenofibrate (FF(20); B), and high-dose fenofibrate (FF(100); C). Increased expression of VEGF was observed in Müller cells (A and B). Rats treated with high-dose fenofibrate showed faint expression of VEGF in Müller cells and a marked decrease in ocular infiltrative cells (C). The bar represents 50 µm.

Effects of fenofibrate on systemic immune responses

On day 28 post-immunization, the ears of the rats were challenged with S-Ag peptide. The rats treated with high-dose fenofibrate exhibited significant inhibition of ear swelling 48 h after the S-Ag peptide challenge compared with the vehicle-treated rats (Figure 5).

Figure 5

Delayed-type hypersensitivity responses to S-antigen peptide. The delayed-type hypersensitivity (DTH) responses to the S-antigen (S-Ag) peptide in rats with experimental autoimmune uveoretinitis (EAU) treated with vehicle (n=10), low-dose fenofibrate (FF(20)), and high-dose fenofibrate (FF(100)) were 4.32±0.92, 3.69±0.82, and 2.15±0.49 mm, respectively. Rats treated with a high dose of fenofibrate exhibited significant inhibition of ear swelling 48 h after S-Ag peptide challenge compared with the vehicle- or low-dose fenofibrate-treated rats. The results are presented as the mean ± standard deviation (SD). *p<0.0001 versus vehicle-treated group, **p=0.0003 versus low-dose fenofibrate-treated group.

The lymphocyte proliferation test (Figure 6) demonstrated that the high dose of fenofibrate induced a decrease in lymphocyte proliferation in the presence of 25 μg/ml S-Ag peptide compared with the vehicle-treated rats.

Figure 6

Proliferative response to the S-antigen peptide in lymph node cells collected from rats treated with vehicle (n=4), low-dose fenofibrate (20 mg/kg, n=4), and high-dose fenofibrate (100 mg/kg, n=4) on day 28 post-immunization. The lymphocyte proliferation test demonstrated that high-dose fenofibrate treatment induced a decrease in lymphocyte proliferation in the presence of 25 mg/ml S-antigen (S-Ag) peptide compared with vehicle-treated groups. *p=0.0008 versus vehicle-treated group.

Discussion

In the present study, our data revealed that oral fenofibrate inhibited the course of the disease in an animal model of uveoretinitis, due to anti-inflammatory and immunomodulatory effects, even though the administration of the drugs was initiated after disease induction. Clinical and histopathological data showed that administration of fenofibrate resulted in a significant anti-inflammatory effect. In addition, the DTH and LNC proliferation data demonstrated that fenofibrate exhibited immunomodulatory effects in EAU. Thus, fenofibrate had a therapeutic effect with partial preservation of the retinal photoreceptors.

PPARα and RXR agonists inhibit microglial and astrocyte production of nitric oxide (NO), IL-1β, tumor necrosis factor-α (TNF-α), IL-6, and monocyte chemoattractant protein-1 (MCP-1) in EAE, an animal model of multiple sclerosis [22,23]. PPARα-deficient mice have abnormally prolonged responses to inflammatory stimuli such as arachidonic acid and leukotrienes [24], and expression of IL-6, vascular cell adhesion molecule (VCAM), and cyclooxygenase-2 in response to cytokine activation can be inhibited by PPARα ligands [25]. PPARα ligands have also been shown to decrease NF-κB activation and IL-12 and IL-6 production in aged mice [26], and may inhibit functional expression of NF-κB, in part by augmenting expression of IκBα [27]. PPARα agonists may mediate protection against EAE in part by repressing transcription factors such as NF-κB and T-bet, which regulate Th1 or Th17 inflammatory genes, as demonstrated for PPARs [16,28-30].

IL-6 and IL-17 are likely to play major roles in the pathogenesis of EAU [31,32]. IL-6 is required for differentiation of Th17 cells, a recently discovered IL-17-producing helper CD4+ T-cell subset [33,34]. Differentiated Th17 cells may recruit inflammatory cells into the retina, and these cells then produce proinflammatory cytokines and chemokines [32]. In this study, we found that fenofibrate inhibited production of increased IL-6 and IL-17 in EAU rats. These cytokines contribute to the development of EAU and retinal degeneration secondary to inflammation, and inhibition of their production may offer an important advantage for visual outcome.

We also found that fenofibrate inhibits Müller glia production of VEGF in EAU. VEGF is an inflammatory cytokine that induces ocular neovascularization and vascular hyperpermeability, which result in retinal or choroidal neovascularization (CNV) secondary to uveitis and uveitic edema. Anti-VEGF therapy has efficacy for CNV secondary to uveitis [35-38]. Fine et al. [39] found increased VEGF levels in the aqueous humor of patients with uveitic cystoid macular edema (CME), and Vinores et al. [40] showed that VEGF expression is upregulated in the inner retina of EAU rats. In the current study, we showed that fenofibrate inhibited the increase in VEGF expression in EAU rats. Since CNV and CME secondary to uveitis cause significant visual impairment, VEGF expression decreased by fenofibrate may result in a significant improvement in vision.

Oral administration of fenofibrate (100 mg/kg) after disease induction ameliorated the disease of EAU in Lewis rats. However, the dose for such effects is far greater than that for controlling hypercholesterolemia in adults. In a recent clinical study, fenofibrate exhibited an anti-inflammatory effect for rheumatoid arthritis at a dose of 145 mg/day [41]. Although the human therapeutic dose of fenofibrate for inhibiting intraocular inflammation has still not been determined, the current study may suggest that the higher dose should be used in humans for inhibiting intraocular inflammation.

The results of this study establish that EAU is ameliorated by administration of fenofibrate. This finding is important because it suggests that PPARα agonists could be used clinically for treatment of immune-mediated inflammatory diseases such as autoimmune uveitis. PPARα agonists are a class of drugs with a favorable safety profile that could improve the quality of life of patients with autoimmune uveitis if proven to be effective in reducing disease severity. The suppressive effect of oral fenofibrate on EAU suggests that fenofibrate may be an effective therapy for autoimmune uveitis. The insights gained from dissecting the mechanism of action of fenofibrate in EAU could also be used for development of new drugs with fewer side effects for treatment of immune-mediated inflammatory diseases.