January 2011
Volume 52, Issue 1
Free
Immunology and Microbiology  |   January 2011
Dynamic Changes of microRNAs in the Eye during the Development of Experimental Autoimmune Uveoretinitis
Author Affiliations & Notes
  • Waka Ishida
    From the Department of Ophthalmology and
  • Ken Fukuda
    From the Department of Ophthalmology and
  • Takuma Higuchi
    the Laboratory of Molecular Biology, Science Research Center, Kochi Medical School, Nankoku, Japan.
  • Mina Kajisako
    From the Department of Ophthalmology and
  • Shuji Sakamoto
    the Laboratory of Molecular Biology, Science Research Center, Kochi Medical School, Nankoku, Japan.
  • Atsuki Fukushima
    From the Department of Ophthalmology and
  • Corresponding author: Atsuki Fukushima, Department of Ophthalmology, Kochi Medical School, Kohasu, Oko-cho, Nankoku-city 783-8505, Japan; fukusima@kochi-u.ac.jp
Investigative Ophthalmology & Visual Science January 2011, Vol.52, 611-617. doi:10.1167/iovs.10-6115
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      Waka Ishida, Ken Fukuda, Takuma Higuchi, Mina Kajisako, Shuji Sakamoto, Atsuki Fukushima; Dynamic Changes of microRNAs in the Eye during the Development of Experimental Autoimmune Uveoretinitis. Invest. Ophthalmol. Vis. Sci. 2011;52(1):611-617. doi: 10.1167/iovs.10-6115.

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      © 2015 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose.: Interleukin (IL)-17–producing Th17 cells play a crucial role in the development of experimental autoimmune uveoretinitis (EAU). Recent studies revealed that the production of cytokines such as IL-17 is controlled by microRNA (miRNA). Here the authors investigated the expression of miRNA in the eye during the development of EAU.

Methods.: To induce EAU, B10.RIII mice were injected with interphotoreceptor retinoid binding protein peptide 161–180 with adjuvants. Control mice received adjuvants alone. Seven, 14, 21, or 28 days after immunization, eyes were harvested for histologic analysis, miRNA array, quantitative real-time polymerase chain reaction (qRT-PCR) for cytokines and miRNAs, and in situ hybridization for miRNAs. Expression levels of cytokines and miRNAs were compared with control mice.

Results.: No histologic changes were observed in eyes collected at day 7. At day 14, EAU was most severe, and thereafter retinal structure was gradually destroyed. Retinal inflammation was not observed in control mice. IL-17A and IL-17F were significantly higher in the eyes of EAU-induced mice at day 7. Array analysis followed by qRT-PCR revealed that in EAU-induced mice, miRNA-142-5p and miRNA-21 were significantly higher, whereas miRNA-182 was significantly lower. These changes could be detected 7 days after EAU induction. In situ hybridization analysis for these miRNAs confirmed qRT-PCR data.

Conclusions.: Expression changes in three miRNAs could be detected in the eye before histologic EAU. Kinetic changes of these miRNAs in the eye paralleled those of IL-17. The possibility that miRNAs can affect IL-17 suggests that miRNAs in the retina regulate the development of EAU.

Experimental autoimmune uveoretinitis (EAU) has been widely used as a model for forms of human uveitis such as Vogt-Koyanagi Harada disease and Behçet's disease. 1 Many studies have demonstrated that interferon (IFN)-γ–producing Th1 cells play a crucial role in the development of EAU. 2 Paradoxical findings on the role of Th1 cells in EAU have been reported. Interleukin (IL)-12 was reported to downregulate EAU 3 ; however, treatment with anti–IFN-γ antibody was reported to exacerbate the severity of EAU. 4 Recently, IL-17–producing Th17 cells were identified as playing a role in organ-specific autoimmune diseases such as collagen-induced arthritis 5 and experimental autoimmune encephalomyelitis (EAE). 6 More recently, Th17 cells were demonstrated to play a critical role in the development of EAU. 7 Importantly, IFN-γ inhibits Th17 development. 8 These findings resolve the question of why IFN-γ suppression exacerbates EAU, a Th1-mediated disease. Furthermore, the Th17/Th1-mediated cytokine network involved in the development of EAU appears to be clarified. 
In the demonstration that IL-17 played a crucial role in the development of EAU, 7 trace amounts of IL-17A transcript were observed in the retinas of EAU mice at day 7. Thus, IL-17A may participate in the development of EAU in the retina at day 7, when histologic EAU is not yet detectable. The IL-17 family is composed of seven ligands and five receptors. 9 IL-17F, a ligand, is homologous (∼60%) to IL-17A. Both IL-17A and IL-17F, which are produced by Th17 cells, exist as homodimers. 10 Recently, IL-17A and IL-17F were also shown to form a heterodimeric cytokine termed IL-17A/F. 11,12 Thus, in addition to IL-17A, IL-17F may play a role in the development of EAU in the retina. 
MicroRNAs (miRNAs) are endogenous, noncoding RNAs that play a crucial role in regulating gene expression in eukaryotes and affect a large number of biological processes, including cell differentiation and development. 13 Recent studies suggest that, in addition to their more recognized role in development, miRNAs play an important role in immunity and autoimmunity. 14,15 miRNAs function in shaping immunity by regulating the repertoire of genes expressed in immune cells and the magnitude and duration of responses to specific pathogens. 16,17 Expression profiling has shown distinct patterns of miRNA expression in different hematopoietic cell lineages, 18 and single miRNAs can have substantial effects in regulating immune responses. 19 For example, miRNA-155 knockdown in mice results in multiple defects in adaptive immunity and enhances IL-4 production. 20,21 With regard to IL-17, in vivo silencing of miRNA-326 resulted in fewer Th17 cells and mild EAE, and its overexpression led to more Th17 cells and severe EAE. 22 Furthermore, expression of miRNA-326 was highly correlated with disease severity in patients with multiple sclerosis and mice with EAE. 22 Thus, cytokine production and associated disease conditions are controlled by miRNAs. 23 However, no studies to date have investigated the participation of miRNA in the development of EAU. Therefore, we sought to investigate the expression of miRNA in the retinas of EAU-developing mice. 
Materials and Methods
Mice and Reagents
B10.RIII mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and were housed under standard (specific pathogen-free) conditions at the Kochi Medical School animal facility. All animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Interphotoreceptor retinoid binding protein (IRBP) 161–180 (SGIPYIISYLHPGNTILHVD) was purchased from Scrum Inc. (Tokyo, Japan) by consignment synthesis. HPLC purity of the peptide was greater than 95%. Complete Freund's adjuvant (CFA) containing 1.0 mg/mL Mycobacterium tuberculosis was obtained from Difco (Baltimore, MD), and pertussis toxin (PTX) was obtained from Sigma-Aldrich (St. Louis, MO). 
Immunization
For induction of EAU, mice (8–12 weeks of age) were immunized subcutaneously with an emulsion (200 μL) containing 100 μg IRBP peptide 161–180 in CFA. PTX (1.0 μg) was concurrently injected intraperitoneally as an additional adjuvant. Control groups of mice were injected subcutaneously with an emulsion of 100 μL PBS and 100 μL CFA, together with intraperitoneal injection of PTX. 
Histopathologic Evaluation
Eyes were enucleated from control and EAU-developing mice on days 7, 14, 21, and 28 after IRBP immunization and fixed for 48 hours in 4% buffered paraformaldehyde until processing. Eyes from naive mice were also collected. Fixed and dehydrated tissue was embedded in paraffin, and 2-μm sections were stained by standard hematoxylin and eosin. The intensity of EAU was scored from 0 to 4 in a blinded manner according to the histopathologic grading described previously for murine EAU. 24  
RNA Extraction and Microarray Experiments
Eyeballs were collected from EAU-developing (day 14) or naive mice, and corneas and lenses were removed. Total RNA was isolated from these ocular tissues using reagent (Isogen; Nippon Gene, Tokyo, Japan) and labeled with Hy3 (sample) and Hy5 (universal reference) dye using a labeling kit (miRCURY LNA microRNA Power Labeling Kit; Exiqon, Vedbaek, Denmark) according to the manufacturer's instructions, and the labeled RNA molecules were hybridized (miRCURY LNA Arrays, version 11.0; Exiqon). A DNA microarray scanner and Feature Extraction software (Agilent Technologies, Santa Clara, CA) were used to analyze images. Arithmetic mean ± SE of triplicate experiments were calculated for each group, and fold changes were measured. Each miRNA signal was transformed to logarithm base 2, and two-sample t-test was conducted. miRNAs with a significance value P < 0.05 and fold change values >2 were considered differentially expressed between naive and EAU-developing eyes. Raw data from the microarray study are deposited in the Gene Expression Omnibus (GEO) database, accession no. GSE22617
Quantitative Assessment of Cytokine and miRNA Levels
Levels of cytokine (IFN-γ, IL-4, IL-12p35, IL-17A, and IL-17F) and miRNA (miRNA-21, miRNA-142-5p, miRNA-182, and miRNA-197) expression in ocular tissues were measured quantitatively by using microRNA assay (Taq-Man; Applied Biosystems, Carlsbad, CA) according to the manufacturer's protocol and were assayed on a sequence detection system (ABI 7000; Applied Biosystems). Normalization was performed with hypoxanthine-guanine phosphoribosyltransferase (HPRT) and snoRNA202 for cytokines and miRNA, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in triplicate, including no-template controls. Relative expression was calculated using the comparative cycle threshold method. 
In Situ Hybridization for miRNA-21, miRNA-142-5p, and miRNA-182
Eyes from EAU-developing (days 7, 14, 21, and 28) and naive mice were embedded in paraffin, and 2-μm sections were cut with a microtome. In situ hybridization was performed according to the manufacturer's protocols (Exiqon). Briefly, slides were prehybridized for 2 hours in a solution of 50% formamide, 5× SSC, 0.1% Tween, 9.2 mM citric acid, 50 μg/mL heparin, and 500 μg/mL yeast RNA. Slides were hybridized overnight at 57°C, with 20 nM digoxigenin-labeled probe per slide, in a humidified chamber. Locked nucleic acid (LNA)-modified oligonucleotide probes (Exiqon) specific for miRNA-21, -142-5p, -182, and a negative control (scramble) were labeled at the 5′ end with digoxigenin and used for hybridization. The hybridized probes were detected using anti–digoxigenin antibodies conjugated to alkaline phosphatase (Roche, Mannheim, Germany), and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Roche) was used as the substrate. After three washings at 57°C, the sections were counterstained with stain solution (Kernechtrot; Muto Chemical, Tokyo, Japan). 
Statistical Analysis
The severity of EAU grading and the expression levels of cytokines and miRNAs evaluated by qRT-PCR in each group were analyzed by Kruskal-Wallis test with the Bonferroni/Dunn test. To compare the two groups (EAU group immunized with IRBP peptide in adjuvant, and control group immunized with adjuvant alone) at each time point (days 7, 14, 21, 28), the Mann-Whitney U test was used because data were not parametrically distributed. P < 0.05 was regarded as significantly different. 
Results
Kinetic Changes of Cytokines in the Eyes during the Development of EAU
To investigate the kinetic changes of cytokines expressed in ocular tissue, eyes were harvested from EAU-developing mice at different time points. As a control, eyes were collected from mice immunized with adjuvant alone. Corneas and lenses were removed, and the residual parts of the eyes were subjected to qRT-PCR analysis to determine the levels of IFN-γ, IL-4, IL-12p35, IL-17A, and IL-17F. The levels of each cytokine at each time point were compared with those of naive animals. In EAU-developing eyes, the upregulation of IL-17A and IL-17F began to be noted at day 7, though it was not significant. Significant upregulation of IFN-γ (Fig. 1A), IL-17A (Fig. 1C), and IL-17F (Fig. 1D) was observed 14 days after EAU induction. Significant upregulation of mRNA expression of these three cytokines was transient, except for a significant upregulation of IL-17F at day 28 (Figs. 1A, 1C, 1D). In contrast, IL-12p35 was suppressed after EAU induction, yet significant differences in expression were not detected at any tested time points (Fig. 1B). IL-4 was not significantly affected by EAU induction (data not shown). In contrast to EAU-developing mice, the levels of all tested cytokines did not differ at any tested time points in control eyes from mice immunized with adjuvants alone (Figs. 1A–D). Comparison between EAU-developing and control eyes revealed that the significant difference regarding IL-17A and IL-17F began to be detectable at day 7. With regard to IFN-γ and IL-12p35, significant differences started to be noted at days 14 and 21, respectively. Significant differences persisted up to 28 days after immunization, except for IL-17F at days 21 and 28. Based on the data that the significant difference between EAU-developing and control mice was not noted in IFN-γ at day 7 of EAU, it could be considered that IL-17A and IL-17F promote EAU and that IFN-γ subsequently participates in sustaining and augmenting EAU. 
Figure 1.
 
qRT-PCR analysis of cytokines in the eye. B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants (filled circles; n = 3 at each time point). As a control, B10.RIII mice were immunized with adjuvants alone (unfilled circles; n = 3 at each time point). Seven, 14, 21, and 28 days later, eyes were collected, and corneas and lenses were removed. As a background, eyes were collected from naive mice (day 0, n = 3). Residual ocular tissues were subjected to qRT-PCR analysis to measure IFN-γ (A), IL-12p35 (B), IL-17A (C), and IL-17F (D) mRNA levels. Data reflect the mRNA level of each cytokine relative to HPRT and are presented in arbitrary units relative to the level of each cytokine in naive mice (set as 1). **P < 0.01 compared with naive mice. †P < 0.05 compared with the control mice immunized with adjuvants alone.
Figure 1.
 
qRT-PCR analysis of cytokines in the eye. B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants (filled circles; n = 3 at each time point). As a control, B10.RIII mice were immunized with adjuvants alone (unfilled circles; n = 3 at each time point). Seven, 14, 21, and 28 days later, eyes were collected, and corneas and lenses were removed. As a background, eyes were collected from naive mice (day 0, n = 3). Residual ocular tissues were subjected to qRT-PCR analysis to measure IFN-γ (A), IL-12p35 (B), IL-17A (C), and IL-17F (D) mRNA levels. Data reflect the mRNA level of each cytokine relative to HPRT and are presented in arbitrary units relative to the level of each cytokine in naive mice (set as 1). **P < 0.01 compared with naive mice. †P < 0.05 compared with the control mice immunized with adjuvants alone.
miRNA-326 Expression in the Eyes during the Development of EAU
Previous studies of EAU in B10.RIII mice demonstrated that the retina and vasculature remain normal in appearance, with no clinical evidence of disease from days 0 to 10 after immunization. 25,26 Interestingly, compared with the control eyes, IL-17A and IL-17F were significantly upregulated at day 7, when no histologic changes were observed in the retina (Figs. 2A, 2B), leading us to further investigate the mechanism underlying IL-17A and IL-17F expression in the retina at this early time point of EAU. Recent studies have revealed that cytokine expression is tightly and specifically regulated at the protein and mRNA levels by noncoding miRNAs. 27 miRNA-326 was recently demonstrated to regulate IL-17A, promote Th17 differentiation, and increase the severity of EAE. 22 Based on these findings, we examined the kinetic changes of miRNA-326 in the eye. After EAU induction, expression of miRNA-326 increased slightly but not significantly (Fig. 2C). In contrast, miRNA-326 expression in control eyes from mice immunized with adjuvants alone did not change up to 28 days after immunization (Fig. 2C). Comparison between EAU-developing and control eyes demonstrated that significant differences began to be noted at day 7 and continued until 21 days after immunization (Fig. 2C). 
Figure 2.
 
Histologic evaluation of EAU. To induce EAU, B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants (filled circles). As a control, B10.RIII mice were immunized with adjuvants alone (unfilled circles). Seven, 14, 21, and 28 days later, the eyes were collected for histologic analysis (A, B; n = 3–6 at each time point). To evaluate miRNA-326 expression by qRT-PCR, corneas and lenses were removed from the enucleated eyes (C; n = 3 at each time point). As a control, eyes were collected from naive mice (day 0; n = 3). (A) Microphotographs of naive and EAU-developing mice. One representative of each time point is shown. (B) Histologic grading. *P < 0.05 and **P < 0.01 compared with naive mice. (C) Expression level of miRNA-326. Data reflect miRNA-326 levels relative to snoRNA202 and are presented in arbitrary units relative to the level of each cytokine in naive mice (set as 1). **P < 0.01 compared with naive mice. †P < 0.05 and ††P < 0.01 compared with the control mice immunized with adjuvants alone.
Figure 2.
 
Histologic evaluation of EAU. To induce EAU, B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants (filled circles). As a control, B10.RIII mice were immunized with adjuvants alone (unfilled circles). Seven, 14, 21, and 28 days later, the eyes were collected for histologic analysis (A, B; n = 3–6 at each time point). To evaluate miRNA-326 expression by qRT-PCR, corneas and lenses were removed from the enucleated eyes (C; n = 3 at each time point). As a control, eyes were collected from naive mice (day 0; n = 3). (A) Microphotographs of naive and EAU-developing mice. One representative of each time point is shown. (B) Histologic grading. *P < 0.05 and **P < 0.01 compared with naive mice. (C) Expression level of miRNA-326. Data reflect miRNA-326 levels relative to snoRNA202 and are presented in arbitrary units relative to the level of each cytokine in naive mice (set as 1). **P < 0.01 compared with naive mice. †P < 0.05 and ††P < 0.01 compared with the control mice immunized with adjuvants alone.
miRNA Expression Profile in the Eye
Although expression levels of miRNA-326 were significantly different between EAU-developing and control eyes, the fold change of upregulation was small. Therefore, we then examined the expression of various miRNAs by using the LNA array (miRCURY; Exiqon). Expression of miRNA in the eye was compared between naive mice and EAU-developing mice at day 14 after EAU induction. Ten of 618 total examined miRNAs were identified as having at least twofold increases or decreases in expression (Table 1). miRNA-466d-5p, miRNA-669f, miRNA-881, miRNA-142-5p, and miRNA-21 were significantly upregulated in EAU eyes compared with naive controls. miRNA-197, miRNA-182, miRNA-183, miRNA-96, and let-7d were significantly downregulated (Table 1). To confirm the reliability of microarray analysis, qRT-PCR was conducted for four selected miRNAs that showed threefold differences in expression level compared with naive controls. qRT-PCR analysis confirmed that miRNA-142-5p and miRNA-21 expression was significantly higher, and miRNA-182 expression was significantly lower, than in controls (Fig. 3); however, no significant difference in miRNA-197 expression was observed (Fig. 3). Therefore, hereafter, we focused on these three miRNAs (miRNA-21, -142-5p, and -182). 
Table 1.
 
microRNA Expression Profile in the Eye
Table 1.
 
microRNA Expression Profile in the Eye
Gene Name* Naive Average† EAU Average† P * EAU/Naive*
miRNA-197 1.736402 0.441444 0.027154 0.254229
miRNA-182 1.784637 0.581616 0.009966 0.325902
miRNA-183 2.413043 0.812685 0.034972 0.336789
miRNA-96 1.936937 0.779001 0.010279 0.402182
let-7d 2.276606 1.031004 0.021998 0.452869
miRNA-466d-5p 1.011287 2.113535 0.035577 2.089945
miRNA-669f 0.7819 1.662853 0.044749 2.126683
miRNA-881 0.805901 1.722513 0.021614 2.137375
miRNA-142–5p 0.384794 1.683884 0.046128 4.376071
miRNA-21 0.311684 2.628999 0.037968 8.434814
Figure 3.
 
qRT-PCR analysis of miRNA expression in the eye. By microarray analysis, 10 of 618 total examined miRNAs were identified as having at least twofold increases or decreases in expression level compared with control (Table 1). To confirm the reliability of microarray analysis, B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants (n = 3). Fourteen days later, the eyes were collected, and the corneas and lenses were removed for qRT-PCR assay. As a control, eyes were collected from naive mice (n = 3). We selected four miRNAs (miRNA-21, miRNA-142-5p, miRNA-182, and miRNA-197) that showed threefold differences in expression between EAU-developing and control mice in the microarray analysis. Data reflect the expression level of each miRNA relative to snoRNA202 and are presented in arbitrary units relative to the level of each miRNA in naive mice (set as 1). Unfilled bars: naive mice; filled bars: EAU-developing mice. *P < 0.05; **P < 0.01.
Figure 3.
 
qRT-PCR analysis of miRNA expression in the eye. By microarray analysis, 10 of 618 total examined miRNAs were identified as having at least twofold increases or decreases in expression level compared with control (Table 1). To confirm the reliability of microarray analysis, B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants (n = 3). Fourteen days later, the eyes were collected, and the corneas and lenses were removed for qRT-PCR assay. As a control, eyes were collected from naive mice (n = 3). We selected four miRNAs (miRNA-21, miRNA-142-5p, miRNA-182, and miRNA-197) that showed threefold differences in expression between EAU-developing and control mice in the microarray analysis. Data reflect the expression level of each miRNA relative to snoRNA202 and are presented in arbitrary units relative to the level of each miRNA in naive mice (set as 1). Unfilled bars: naive mice; filled bars: EAU-developing mice. *P < 0.05; **P < 0.01.
Kinetic Changes of miRNA Expression during the Development of EAU
Next, to reveal the kinetic changes of miRNA expression, the eyes of naive and EAU-developing mice (7, 14, 21, and 28 days after induction) were subjected to qRT-PCR assays. Significant upregulation of miRNA-21 (Fig. 4A) and significant downregulation of miRNA-182 (Fig. 4C) were noted 14 days after EAU induction. Significant upregulation of miRNA-142-5p was observed 21 days after EAU induction (Fig. 4B). Importantly, miRNA expression levels in control eyes from mice immunized with adjuvants alone did not change significantly. Similar to IL-17A and IL-17F, significant differences concerning miRNA-21 (Fig. 4A) and miRNA-142-5p (Fig. 4B) began to be noted at day 7 between EAU-developing and control eyes. With regard to miRNA-182, significant differences began to be noted at day 14 (Fig. 4C). These significant differences persisted up to 28 days after immunization (Fig. 4). 
Figure 4.
 
Kinetic changes of miRNA expression in EAU-developing eyes. B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants (filled circles; n = 3 at each time point). As a control, B10.RIII mice were immunized with adjuvants alone (unfilled circles; n = 3 at each time point). Seven, 14, 21, and 28 days later, the eyes were collected and corneas and lenses were removed from the enucleated eyes for qRT-PCR assays to evaluate the expression levels of miRNA-21, miRNA-142-5p, and miRNA-182. The expression level of each miRNA was compared with that of naive mice (day 0; n = 3). Data reflect the expression level of each miRNA relative to snoRNA202 and are presented in arbitrary units relative to the level of each miRNA in naive mice (set as 1). **P < 0.01 compared with naive mice. †P < 0.05 compared with control mice immunized with adjuvants alone.
Figure 4.
 
Kinetic changes of miRNA expression in EAU-developing eyes. B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants (filled circles; n = 3 at each time point). As a control, B10.RIII mice were immunized with adjuvants alone (unfilled circles; n = 3 at each time point). Seven, 14, 21, and 28 days later, the eyes were collected and corneas and lenses were removed from the enucleated eyes for qRT-PCR assays to evaluate the expression levels of miRNA-21, miRNA-142-5p, and miRNA-182. The expression level of each miRNA was compared with that of naive mice (day 0; n = 3). Data reflect the expression level of each miRNA relative to snoRNA202 and are presented in arbitrary units relative to the level of each miRNA in naive mice (set as 1). **P < 0.01 compared with naive mice. †P < 0.05 compared with control mice immunized with adjuvants alone.
To confirm the kinetic changes of miRNA expression in the eye, in situ hybridization assays were conducted. Control probe (scramble) was not hybridized to the retina, regardless of EAU induction, because purple staining was not observed (Fig. 5). As purple staining was observed strikingly in the retina, especially in the outer granular layer, miRNA-182 was abundantly expressed in retinal cells in naive mice, as previously reported, 28 and gradually decreased as EAU progressed (Fig. 5). In contrast, as the intensities of purple staining became distinguishable between day 0 and day 7, the expression of miRNA-21 (Fig. 5) and miRNA-142-5p (Fig. 5) increased by EAU induction. The strong upregulation of these two miRNAs at later time points (days 14, 21, and 28) demonstrated by qRT-PCR (Figs. 4A, 4B) might have been due to the relative increase of inflammatory cells compared with ocular cells. These two miRNAs are known to be abundantly expressed in inflammatory cells, and the total volume of retinal cells decreased as EAU progressed (Fig. 2A). 
Figure 5.
 
In situ hybridization of miRNA in EAU-developing eyes. B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants. Seven, 14, 21, and 28 days later (n = 3 at each time point), eyes were collected and embedded in paraffin, and 2-μm sections were cut with a microtome. As a control, eyes were collected from naive mice (day 0; n = 3). Slides were then hybridized with LNA-modified oligonucleotide probes specific for miRNA-21, -142-5p, -182, and a negative control (Scramble). One representative of each time point is shown. Purple: positive signal. Scale bar, 50 μm.
Figure 5.
 
In situ hybridization of miRNA in EAU-developing eyes. B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants. Seven, 14, 21, and 28 days later (n = 3 at each time point), eyes were collected and embedded in paraffin, and 2-μm sections were cut with a microtome. As a control, eyes were collected from naive mice (day 0; n = 3). Slides were then hybridized with LNA-modified oligonucleotide probes specific for miRNA-21, -142-5p, -182, and a negative control (Scramble). One representative of each time point is shown. Purple: positive signal. Scale bar, 50 μm.
Discussion
In this study, we demonstrated that the expression of IL-17A, IL-17F, and IFN-γ mRNA is upregulated in the eye during the development of EAU, whereas IL-12p35 mRNA is downregulated. Interestingly, IL-17A and IL-17F in eyes with EAU were significantly higher than in control eyes at day 7, when histologic EAU was not yet observed. These results provide a possibility that these cytokines can be produced by retinal cells before the onset of retinal inflammation. However, a recent paper demonstrated that a small number of inflammatory cells infiltrated the retina as early as 5 days after EAU induction, when histologic EAU was not yet detectable. 29 The infiltrating cell number into the retina was stable up to day 10 of EAU. 29 Thus, the source of these cytokines in the eye at day 7 can be attributed to both retinal cells and a small number of retina-infiltrating cells. 
Recently, miRNAs have emerged as important regulators of gene expression in the immune system by functioning as endogenous inhibitors of translational processes. 30 In EAE, whose mechanism of development closely resembles that of EAU, 7 miRNA-326 was found to regulate Th17 differentiation, which is a crucial event for the development of EAE. 22 Consistent with this report, and compared with control eyes, miRNA-326 was significantly upregulated in the eye after the induction of EAU. However, the fold change was small compared with the data of EAE; the reason for this differential contribution of miRNA-326 between EAE and EAU remains unclear and must be clarified in the future. Therefore, we examined the possibility that miRNAs other than miRNA-326 are upregulated or downregulated in the eye during the development of EAU. 
Microarray experiments followed by qRT-PCR analysis demonstrated that miRNA-182 was significantly suppressed, whereas miRNA-142-5p and miRNA-21 were significantly upregulated in the eye during the development of EAU. These changes were further confirmed by in situ hybridization analysis. Changes in expression levels of miRNA-142-5p and miRNA-21 were observed at day 7, when significant upregulation of IL-17 was also noted but histologic EAU had not yet occurred. Importantly, immunohistochemical analysis demonstrated that no CD4+ or CD11b+ cells were detected in the retina at day 7 of EAU (data not shown). On the contrary, in situ hybridization analysis of miRNA-21 and miRNA-142-5p demonstrated that apparently more purple dots were detected in the retina at day 7 of EAU-developing mice than naive mice. Therefore, changes in miRNA levels in the eye at day 7 can be attributed to miRNAs derived from the retina rather than miRNAs derived from EAU-induced inflammatory cells. 
To investigate the possible involvement of these miRNAs in IL-17 expression, we searched target miRNA for IL-17A and IL-17F by using TargetScan (http://www.targetscan.org/). miRNA-182 has sites with a lower probability of preferential conservation for IL-17A. Thus, it is conceivable that the downregulation of miRNA-182 plays a role in the upregulation of IL-17A. With regard to miRNA-21, a previous report demonstrated that the introduction of pre–miRNA-21 inhibited cellular expression of a reporter vector harboring the 3′-untranslated region of IL-12p35, indicating that miRNA-21 targets IL-12p35. 27 Indeed, IL-12p35 expression was suppressed in EAU-developing eyes. IFN-γ produced by Th1 cells was reported to inhibit the development of Th17 cells, 8 and IFN-γ production in Th1 cells is promoted by IL-12. 31 Taken together, the suppression of IL-12p35 production by the upregulation of miRNA-21 may promote IL-17 production. 
TargetScan analysis showed that miRNA-142-5p does not have sites of preferential conservation for IL-17. As reported previously, miRNA-326 promoted Th17 differentiation not by a direct effect but by targeting the transcription factor Ets-1, a negative regulator of Th-17 differentiation. 22 Thus, it may be possible that miRNA-142-5p indirectly affects IL-17 production. Importantly, miRNA-142-5p is highly expressed in hematopoietic cells. 32 Therefore, upregulation of this miRNA in the eye later than day 14 of EAU can be attributed to inflammatory cells infiltrating the retina. TargetScan analysis showed that miRNA-142-5p has sites with a lower probability of preferential conservation for PKCα, 33 interphotoreceptor matrix proteoglycan 1, 34 and Calbindin D-28 kDa, 35 which are expressed in retinal cells such as rod bipolar cells and horizontal cells. Thus, the change of miRNA-142-5p levels in the EAU-developing eye at day 7 may be attributed to changes in these retinal constitutive cells. 
The miRNA cluster, including miRNA-96, miRNA-182, and miRNA-183, is located in mouse chromosome 6qA3, with conservation of synteny to human chromosome 7q32.3. 36 A previous report demonstrated that expression of this cluster is restricted to sensory neurons of the retina, inner ear, olfactory epithelium, taste buds, and dorsal root ganglia. 37 Our data that miRNA-182 expression in the eye was downregulated by EAU induction may be related to the destruction of retinal structure. Indeed, miRNA-96 and miRNA-183 were also significantly downregulated by the induction of EAU (Table 1 and data not shown, confirmed by qRT-PCR). Thus, we should pay attention to the possibility that fluctuation of miRNA expression can be an indirect change. 
Given that few papers are available regarding the relationship between miRNA and autoimmune diseases, our data presented here provide some valuable information regarding the regulation and control of autoimmune diseases. Furthermore, we found that the expression of miRNA changes in the eye before histologically detectable inflammation. Taken together, our findings indicate that retinal cells express miRNAs and may reflect pathologic changes before the onset of inflammatory conditions. Because this study lacks direct evidence of miRNA function in the development of EAU, further studies using gene transfer methods are needed to clarify how miRNA plays a role in the development of EAU. 
Footnotes
 Supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (AF).
Footnotes
 Disclosure: W. Ishida, None; K. Fukuda, None; T. Higuchi, None; M. Kajisako, None; S. Sakamoto, None; A. Fukushima, None
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Figure 1.
 
qRT-PCR analysis of cytokines in the eye. B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants (filled circles; n = 3 at each time point). As a control, B10.RIII mice were immunized with adjuvants alone (unfilled circles; n = 3 at each time point). Seven, 14, 21, and 28 days later, eyes were collected, and corneas and lenses were removed. As a background, eyes were collected from naive mice (day 0, n = 3). Residual ocular tissues were subjected to qRT-PCR analysis to measure IFN-γ (A), IL-12p35 (B), IL-17A (C), and IL-17F (D) mRNA levels. Data reflect the mRNA level of each cytokine relative to HPRT and are presented in arbitrary units relative to the level of each cytokine in naive mice (set as 1). **P < 0.01 compared with naive mice. †P < 0.05 compared with the control mice immunized with adjuvants alone.
Figure 1.
 
qRT-PCR analysis of cytokines in the eye. B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants (filled circles; n = 3 at each time point). As a control, B10.RIII mice were immunized with adjuvants alone (unfilled circles; n = 3 at each time point). Seven, 14, 21, and 28 days later, eyes were collected, and corneas and lenses were removed. As a background, eyes were collected from naive mice (day 0, n = 3). Residual ocular tissues were subjected to qRT-PCR analysis to measure IFN-γ (A), IL-12p35 (B), IL-17A (C), and IL-17F (D) mRNA levels. Data reflect the mRNA level of each cytokine relative to HPRT and are presented in arbitrary units relative to the level of each cytokine in naive mice (set as 1). **P < 0.01 compared with naive mice. †P < 0.05 compared with the control mice immunized with adjuvants alone.
Figure 2.
 
Histologic evaluation of EAU. To induce EAU, B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants (filled circles). As a control, B10.RIII mice were immunized with adjuvants alone (unfilled circles). Seven, 14, 21, and 28 days later, the eyes were collected for histologic analysis (A, B; n = 3–6 at each time point). To evaluate miRNA-326 expression by qRT-PCR, corneas and lenses were removed from the enucleated eyes (C; n = 3 at each time point). As a control, eyes were collected from naive mice (day 0; n = 3). (A) Microphotographs of naive and EAU-developing mice. One representative of each time point is shown. (B) Histologic grading. *P < 0.05 and **P < 0.01 compared with naive mice. (C) Expression level of miRNA-326. Data reflect miRNA-326 levels relative to snoRNA202 and are presented in arbitrary units relative to the level of each cytokine in naive mice (set as 1). **P < 0.01 compared with naive mice. †P < 0.05 and ††P < 0.01 compared with the control mice immunized with adjuvants alone.
Figure 2.
 
Histologic evaluation of EAU. To induce EAU, B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants (filled circles). As a control, B10.RIII mice were immunized with adjuvants alone (unfilled circles). Seven, 14, 21, and 28 days later, the eyes were collected for histologic analysis (A, B; n = 3–6 at each time point). To evaluate miRNA-326 expression by qRT-PCR, corneas and lenses were removed from the enucleated eyes (C; n = 3 at each time point). As a control, eyes were collected from naive mice (day 0; n = 3). (A) Microphotographs of naive and EAU-developing mice. One representative of each time point is shown. (B) Histologic grading. *P < 0.05 and **P < 0.01 compared with naive mice. (C) Expression level of miRNA-326. Data reflect miRNA-326 levels relative to snoRNA202 and are presented in arbitrary units relative to the level of each cytokine in naive mice (set as 1). **P < 0.01 compared with naive mice. †P < 0.05 and ††P < 0.01 compared with the control mice immunized with adjuvants alone.
Figure 3.
 
qRT-PCR analysis of miRNA expression in the eye. By microarray analysis, 10 of 618 total examined miRNAs were identified as having at least twofold increases or decreases in expression level compared with control (Table 1). To confirm the reliability of microarray analysis, B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants (n = 3). Fourteen days later, the eyes were collected, and the corneas and lenses were removed for qRT-PCR assay. As a control, eyes were collected from naive mice (n = 3). We selected four miRNAs (miRNA-21, miRNA-142-5p, miRNA-182, and miRNA-197) that showed threefold differences in expression between EAU-developing and control mice in the microarray analysis. Data reflect the expression level of each miRNA relative to snoRNA202 and are presented in arbitrary units relative to the level of each miRNA in naive mice (set as 1). Unfilled bars: naive mice; filled bars: EAU-developing mice. *P < 0.05; **P < 0.01.
Figure 3.
 
qRT-PCR analysis of miRNA expression in the eye. By microarray analysis, 10 of 618 total examined miRNAs were identified as having at least twofold increases or decreases in expression level compared with control (Table 1). To confirm the reliability of microarray analysis, B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants (n = 3). Fourteen days later, the eyes were collected, and the corneas and lenses were removed for qRT-PCR assay. As a control, eyes were collected from naive mice (n = 3). We selected four miRNAs (miRNA-21, miRNA-142-5p, miRNA-182, and miRNA-197) that showed threefold differences in expression between EAU-developing and control mice in the microarray analysis. Data reflect the expression level of each miRNA relative to snoRNA202 and are presented in arbitrary units relative to the level of each miRNA in naive mice (set as 1). Unfilled bars: naive mice; filled bars: EAU-developing mice. *P < 0.05; **P < 0.01.
Figure 4.
 
Kinetic changes of miRNA expression in EAU-developing eyes. B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants (filled circles; n = 3 at each time point). As a control, B10.RIII mice were immunized with adjuvants alone (unfilled circles; n = 3 at each time point). Seven, 14, 21, and 28 days later, the eyes were collected and corneas and lenses were removed from the enucleated eyes for qRT-PCR assays to evaluate the expression levels of miRNA-21, miRNA-142-5p, and miRNA-182. The expression level of each miRNA was compared with that of naive mice (day 0; n = 3). Data reflect the expression level of each miRNA relative to snoRNA202 and are presented in arbitrary units relative to the level of each miRNA in naive mice (set as 1). **P < 0.01 compared with naive mice. †P < 0.05 compared with control mice immunized with adjuvants alone.
Figure 4.
 
Kinetic changes of miRNA expression in EAU-developing eyes. B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants (filled circles; n = 3 at each time point). As a control, B10.RIII mice were immunized with adjuvants alone (unfilled circles; n = 3 at each time point). Seven, 14, 21, and 28 days later, the eyes were collected and corneas and lenses were removed from the enucleated eyes for qRT-PCR assays to evaluate the expression levels of miRNA-21, miRNA-142-5p, and miRNA-182. The expression level of each miRNA was compared with that of naive mice (day 0; n = 3). Data reflect the expression level of each miRNA relative to snoRNA202 and are presented in arbitrary units relative to the level of each miRNA in naive mice (set as 1). **P < 0.01 compared with naive mice. †P < 0.05 compared with control mice immunized with adjuvants alone.
Figure 5.
 
In situ hybridization of miRNA in EAU-developing eyes. B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants. Seven, 14, 21, and 28 days later (n = 3 at each time point), eyes were collected and embedded in paraffin, and 2-μm sections were cut with a microtome. As a control, eyes were collected from naive mice (day 0; n = 3). Slides were then hybridized with LNA-modified oligonucleotide probes specific for miRNA-21, -142-5p, -182, and a negative control (Scramble). One representative of each time point is shown. Purple: positive signal. Scale bar, 50 μm.
Figure 5.
 
In situ hybridization of miRNA in EAU-developing eyes. B10.RIII mice were immunized with IRBP peptide 161–180 with adjuvants. Seven, 14, 21, and 28 days later (n = 3 at each time point), eyes were collected and embedded in paraffin, and 2-μm sections were cut with a microtome. As a control, eyes were collected from naive mice (day 0; n = 3). Slides were then hybridized with LNA-modified oligonucleotide probes specific for miRNA-21, -142-5p, -182, and a negative control (Scramble). One representative of each time point is shown. Purple: positive signal. Scale bar, 50 μm.
Table 1.
 
microRNA Expression Profile in the Eye
Table 1.
 
microRNA Expression Profile in the Eye
Gene Name* Naive Average† EAU Average† P * EAU/Naive*
miRNA-197 1.736402 0.441444 0.027154 0.254229
miRNA-182 1.784637 0.581616 0.009966 0.325902
miRNA-183 2.413043 0.812685 0.034972 0.336789
miRNA-96 1.936937 0.779001 0.010279 0.402182
let-7d 2.276606 1.031004 0.021998 0.452869
miRNA-466d-5p 1.011287 2.113535 0.035577 2.089945
miRNA-669f 0.7819 1.662853 0.044749 2.126683
miRNA-881 0.805901 1.722513 0.021614 2.137375
miRNA-142–5p 0.384794 1.683884 0.046128 4.376071
miRNA-21 0.311684 2.628999 0.037968 8.434814
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