Biochemistry and Molecular Biology Role of miR-146a in the Regulation of Inflammation in an In Vitro Model of Graves’ Orbitopathy

Sun Young Jang,1,2 Min Kyung Chae,3 Joon H. Lee,4 Eun Jig Lee,5 and Jin Sook Yoon3

1Department of Ophthalmology, Soonchunhyang University Bucheon Hospital, Soonchunhyang University College of Medicine, Bucheon, Korea 2Department of Medicine, Yonsei University Graduate School of Medicine, Seoul 3Department of Ophthalmology, Severance Hospital, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea 4Myung-Gok Eye Research Institute at Kim’s Eye Hospital, Konyang University College of Medicine, Nonsan, Korea 5Department of Endocrinology, Severance Hospital, Institue of Endocrine Research, Yonsei University College of Medicine, Seoul, Korea

Correspondence: Jin Sook Yoon, PURPOSE. To investigate the role of microRNA 146a (miR-146a) in the regulation of Institute of Vision Research, Depart- inflammation in an in vitro model of Graves’ orbitopathy (GO). ment of Ophthalmology, Yonsei Uni- versity College of Medicine, 50 METHODS. The level of miR-146a expression in orbital adipose tissue was compared between Yonsei-ro, Seodaemun-ku, 03722 GO and non-GO by quantitative real-time PCR (qPCR). The effects of interleukin 1b (IL-1b)on Seoul, Korea; miR-146a expression were analyzed in orbital fibroblasts by qPCR. To investigate the [email protected]. molecular mechanism underlying IL-1b–induced miR-146a expression, the effects of Submitted: January 25, 2016 inhibitors of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-jB), mitogen- Accepted: June 25, 2016 activated protein kinase/extracellular signal–regulated kinases (MEK)-1/2, c-Jun N-terminal kinases (JNK)-1/2, p38 MAP kinase, and phosphatidylinositol-4,5-bisphosphate 3-kinase Citation: Jang SY, Chae MK, Lee JH, Lee EJ, Yoon JS. Role of miR-146a in (PI3K) were analyzed. The effects of miR-146a mimics and inhibitors on IL-1b–induced IL-6 the regulation of inflammation in an in release were examined by ELISA and Western blotting. vitro model of Graves’ orbitopathy. RESULTS. The level of miR-146a expression was significantly higher in GO orbital adipose tissue Invest Ophthalmol Vis Sci. than in non-GO (P ¼ 0.032). Interleukin 1b induced a time- and concentration-dependent 2016;57:4027–4034. DOI:10.1167/ increase in miR-146a expression. Interleukin 1b (10ng/mL,16hours)inducedan iovs.16-19213 approximately 17.5-fold increase in miR-146 expression. The increase in miR-146a expression by IL-1b was significantly inhibited by NF-jB, JNK-1/2, and PI3K inhibitors (1.94 6 0.25, 5.28 6 0.34 and 9.73 6 2.32-fold, respectively, P < 0.05 compared with IL-1b–induced miR- 146 expression, independent t-test). Interleukin 1b–induced IL-6 protein production was further decreased by miR-146a mimics, but not by inhibitors of miR-146a.

CONCLUSIONS. MicroRNA 146a was upregulated by inflammatory stress in orbital fibroblasts. Our results indicated that miR-146a had a positive effect on the anti-inflammatory process. MicroRNA 146a may play a role in the regulation of inflammation in orbital fibroblasts, and may participate in the pathogenesis of GO. Keywords: miR-146a, inflammation, Graves’ orbitopathy

raves’ orbitopathy (GO) is an inflammatory autoimmune important regulatory roles by targeting mRNAs for cleavage or G disorder of the orbit. Previous studies have indicated that translational repression.8–10 Thus, they negatively regulate gene the thyroid stimulating hormone (TSH) receptor, which is expression at the posttranscriptional level. Several recent expressed on orbital fibroblasts, is the autoimmune target of studies have shown that inflammatory autoimmune diseases, GO.1–4 Binding of autoantibodies to TSH receptors, expressed such as rheumatoid arthritis (RA),11 systemic lupus erythema- on orbital fibroblasts, activates the T cell–dependent inflam- tosus (SLE),12,13 ulcerative colitis,14 and psoriasis15 have been matory process. Thus, GO is believed to be related to T cell– reported to be associated with altered miRNA expression. Thus, mediated autoimmunity to an antigen present in orbital based on reports that inflammatory autoimmune conditions are fibroblasts. Activated CD4þ T cells secrete IL-1, IFN-c, and related to altered miRNA expression, we postulated that TNF-a, inducing the expression of TSH receptor and CD40 on specific miRNAs may also be associated with GO. the surface of orbital fibroblasts, which promote the secretion Located in the LOC285628 gene on human chromosome 5, of IL-6, 8, fibronectin, type 1 collagen, and glycosaminogly- miRNA 146a (miR-146) is a relatively well known miRNA in cans.5–7 Interaction with CD4þ T cells enhances orbital inflammatory autoimmune diseases.10,16 Studies have indicated fibroblast activation, proliferation, differentiation, and lipid that miR-146a plays an important role in the pathogenesis of accumulation. several autoimmune disorders, such as RA,11,17 SLE,13 osteoar- MicroRNAs (miRNAs) are endogenous, single-stranded, thritis (OA),18 and Sj¨ogren syndrome.19 MicroRNA 146a seems noncoding RNAs, 18 to 24 nucleotides in length that can play to act through inhibition of the nuclear factor kappa-light-chain-

iovs.arvojournals.org j ISSN: 1552-5783 4027

This work is licensed under a Creative Commons Attribution 4.0 International License. Downloaded from iovs.arvojournals.org on 09/28/2021 Role of miR-146a in Graves’ Orbitopathy IOVS j August 2016 j Vol. 57 j No. 10 j 4028

enhancer of activated B cell (NF-jB) pathway by downregula- The data were processed based on the quantile normalization tion of its target genes, such as TNF receptor–associated factor method using commercial software (GeneSpring GX 13.1; 6 (TRAF6) and IL-1 receptor–associated kinase 1 (IRAK1).20 Agilent Technologies). This normalization method aims to This leads to termination or mitigation of an inflammatory achieve a consistent distribution of intensities for each of a set response. Based on this background, we focused on miR-146a of arrays. The normalized and log-transformed intensity values in an inflammatory cellular model of orbital fibroblast were then analyzed using commercial software (Agilent inflammation induced by IL-1b. Technologies). The fold changes in the miRNA expression In this study, we first compare the expression levels of miR- between the GO and non-GO samples were calculated from 146a in orbital adipose tissue between GO and non-GO. Then, the signal values. MicroRNA expression was considered we determine the role of miR-146a in the regulation of significantly different if the fold change exceeded 1.5. Target inflammation in an in vitro model of GO. prediction was performed using a cutoff at the 95% percentile using the TargetScan6.2 database in the public domain (http:// www.targetscan.org/). METHODS Measurement of miR-146a Expression by Subjects and Cell Culture Protocol Quantitative Real-Time PCR Orbital adipose/connective tissue explants were obtained For quantitative real-time PCR (qPCR), the total RNA (1 g) of from 19 GO patients and from 17 age- and sex-matched l each orbital adipose/connective tissue sample obtained from control subjects with no history of GO. All GO patients another five GO patients and five non-GO normal controls was underwent orbital decompression for proptosis correction isolated using a miRNA isolation kit (mirVana; Ambion, Austin, and control subjects underwent cosmetic upper and lower TX, USA), and reverse-transcribed into complementary DNA blepharoplasty. Informed written consent was obtained from using a microRNA reverse transcription kit (TaqMan; Applied all subjects and this study was approved by the Institutional Biosystems, Carlsbad, CA, USA). The resulting cDNA was Review Board of Severance Hospital, Yonsei University amplified using a thermocycler (ABI StepOnePlus Real Time College of Medicine. All GO patients were euthyroid status PCR; Applied Biosystems) with a universal PCR master mix at the time of surgery and had not been treated with steroids (TaqMan No AmpErase UNG; Applied Biosystems) and the or radiation therapy for at least 3 months. recommended PCR conditions for quantitative assessment of Orbital fibroblast cell cultures were performed according to 21–23 gene transcript levels in the tissue samples. All PCRs were the methods described previously. Briefly, for primary cell performed in triplicate. The catalog number of the primers cultures, tissue explants were minced and placed in plastic used was 000468 for miR-146a. RNU6B expression was used culture dishes containing Dulbecco’s modified Eagle’s medium for normalization, and the results were expressed as relative (DMEM):F12 (1:1) (Lonza, Basel, Switzerland), 20% fetal bovine fold changes of threshold cycle (Ct) value relative to the serum (FBS; Life Technologies, Carlsbad, CA, USA), and control group using the 2–DDCt method.24 penicillin–streptomycin (Life Technologies). After orbital fibro- blasts had grown out from the explants, monolayers were passaged serially by gentle treatment with trypsin/EDTA, and Cell Stimulation cells were incubated in DMEM with 10% FBS and antibiotics. Orbital fibroblasts were plated onto 6-well plates for assess- Cell cultures were grown in a humidified 5% CO2 incubator at ment of cytokine release and RNA extraction. Cells were 378C. Cells were stored in liquid N2 until needed and used stimulated in triplicate in DMEM:F12 with the indicated IL-1b between the third and seventh passage. (R&D Systems, Minneapolis, MN, USA) concentration (0, 1, 5, 10, and 20 ng/mL) or with 10 ng/mL of IL-1b for the indicated Microarray Analysis time (0, 3, 6, 16, and 24 hours). To assess the molecular mechanism of IL-1b–induced miR- For the microarray analysis, the total RNA in each orbital 146a expression, the effects of inhibitors of MEK-1/2, JNK-1/2, adipose/connective tissue sample obtained from eight GO p38 MAP kinase, and PI3-K were investigated using GO orbital patients and six non-GO normal controls was extracted fibroblasts. In the present study, we used MEK-1/2 (PD098059; using a commercial reagent (TRI Reagent; Molecular Sigma-Aldrich Corp., St. Louis, MO, USA), JNK-1/2 (SP600125; Research Center, Cincinnati, OH, USA), according to the Sigma-Aldrich Corp.), p38 MAP kinase (SB203580; Sigma- manufacturer’s instructions. The quality and quantity of total Aldrich Corp.), and PI3-K (LY294002; Sigma-Aldrich Corp.). RNA were assessed using a bioanalyzer (Agilent Bioanalyzer To examine the effects on IL-1b–induced miR-146a expression, 2100; Agilent Technologies, Santa Clara, CA, USA). Starting these inhibitors were added at a concentration of 20 lM60 with 250 ng of total RNA, the labeling process began by minutes prior to addition of IL-1b (10 ng/mL). adding a poly(A) tail to each RNA strand using poly(A) To assess the possible involvement of proinflammatory polymerase, followed by ligation of biotin-labeled 3DNA transcription factors, including the NF-jB pathway, the effects dendrimer. Biotinylated RNA strands were hybridized at 488C of preincubation with SC-514 (Calbiochem, La Jolla, CA, USA), for 18 hours on a miRNA array (Affymetrix GeneChip miRNA a selective inhibitor of kappa light polypeptide gene enhancer 4.0 Array; Affymetrix, Santa Clara, CA, USA). The miRNA in B-cells (IjB) kinase-2 inhibitor, and dexamethasone were array (Affymetrix), with 2578 human mature miRNA, was examined.25 Following 1-hour pretreatment with SC-514 (100 washed and stained in an array cartridge system (Affymetrix lM) and dexamethasone (0.1 lM), orbital fibroblasts were Fluidics Station 450; Affymetrix). Amplified fluorescent stimulated with IL-1b (10 ng/mL) and the expression of miR- signals were scanned using a commercial scanner (Affyme- 146a was determined at 16 hours. trix GeneChip Scanner 3000 7G; Affymetrix). The arrays were analyzed using a scanner with associated Transfection With miR-146a Mimics and Inhibitors software (Agilent). The miRNA expression levels were calculated with a commercial console (Expression Console Orbital fibroblasts were transfected with miR-146a mimics, 1.4; Affymetrix). Relative signal intensities for each miRNA inhibitors, and each control according to the respective were generated using the robust multi-array average algorithm. manufacturer’s protocol. Three separate experiments were

Downloaded from iovs.arvojournals.org on 09/28/2021 Role of miR-146a in Graves’ Orbitopathy IOVS j August 2016 j Vol. 57 j No. 10 j 4029

FIGURE 1. MicroRNA expression profiling in GO. The heat map represents the expression value of each specimen (eight GO and six non-GO) using color intensity. Gene names of microRNA, showing a more than 1.5-fold change, are listed to the right. In total, 38 miRNAs were upregulated and 7 microRNAs were downregulated in orbital connective tissue from GO patients, compared with healthy normal controls. Six of the eight GO subjects had higher miR-146a expression level than the mean control value. miR, microRNA; NL, normal.

performed using cells from three different individuals. The to stimulation with 10 ng/mL IL-1b. Supernatants were microRNA 146a mimics were obtained from Ambion/Applied removed at 24 hours and IL-6 levels were determined by ELISA. Biosystems and miR-146a inhibitors were obtained from Exiqon (Vedbaek, Denmark). We used a commercial reagent Western Blotting (Lipofectamine 2000; Life Technologies) as a negative control. Orbital fibroblasts were plated onto six-well plates for The effects of miR-146a mimics on IL-1b–induced IL-6, cyclooxygenase (COX)-2, and intercellular adhesion mole- assessment of cytokine release. Cells were transfected with cule (ICAM)-1 release in GO orbital fibroblasts were analyzed the indicated miR146a mimics and inhibitors at concentrations by Western blotting. Transfected cells were washed with ice- of 0, 10, 30, or 100 nM using commercial reagents (Lipofect- cold PBS, and whole-cell lysates were obtained by incuba- amine RNAiMAX; Life Technologies). tion on ice for 30 minutes in cell lysis buffer (20 mM HEPES, pH 7.2, 10% (vol/vol) glycerol, 10 mM Na3VO4,50mMNaF, Measurement of IL-6 Secreted by ELISA 1 mM phenylmethylsulfonyl fluoride, 0.1 mM dithiothreitol, 1 lg/mL leupeptin, 1 lg/mL pepstatin, and 1% (vol/vol) The effects of miR-146a mimics and inhibitors on IL-1b– Triton X-100). Reagents were purchased from Sigma-Aldrich induced IL-6 release expression were analyzed using a human Corp. Lysates were centrifuged at 12,000g for 10 minutes cytokine ELISA kit (R&D Systems) according to the manufac- and the cell homogenate fractions were stored at 708Cuntil turer’s protocol for three GO and three non-GO orbital use. fibroblasts from different individuals. Protein concentrations were determined by the Bradford Transfected cells were plated into six-well plates and left to assay.21,22 Equal amounts of protein (50 lg) were boiled in adhere overnight. The cells were then starved for 6 hours prior sample buffer and resolved by 10% (wt/vol) SDS-PAGE.

Downloaded from iovs.arvojournals.org on 09/28/2021 Role of miR-146a in Graves’ Orbitopathy IOVS j August 2016 j Vol. 57 j No. 10 j 4030

FIGURE 2. Expression of miR-146a in GO. Expression of miR-146a was FIGURE 4. Effects of inhibitors of MEK-1/2, JNK-1/2, p38 MAP kinase, significantly higher in orbital adipose tissues from GO than from non- PI3-K, NF-jB and dexamethasone on IL-1b–induced miR-146a expres- GO subjects. (*P < 0.05). The results are expressed as the means 6 sion in GO orbital fibroblasts. The increase in miR-146a expression standard deviation of five individual samples and the graphs are induced by IL-1b (17.54 6 3.84-fold) was inhibited by JNK-1/2 (5.28 representative of three independent experiments. 6 0.34-fold, P ¼ 0.030) and PI3K inhibitors (9.73 6 2.32-fold, P ¼ 0.039), but not by MEK-1/2 or p38 MAP kinase inhibitors (*P < 0.05, versus IL-1b–induced miR-146 expression). The level of miR-146a expression was significantly decreased by pretreatment with SC-514 Proteins were transferred onto polyvinylidene difluoride (1.94 6 0.25-fold) and dexamethasone (4.95 6 0.48-fold) compared membranes (Immobilon; Millipore, Billerica, MA, USA). The with the IL-1b–induced miR-146 expression (17.54 6 3.84-fold, P ¼ samples were probed overnight with primary antibodies (IL- 0.019 and 0.029, respectively). The results are expressed as the means 6, COX-2, and ICAM-1) in tris-buffered saline containing 6 standard deviation of three individual samples and the graphs are representative of three independent experiments. Dexa, dexametha- Tween 20 (TBST), and washed three times with TBST. sone; LY, PI3-K inhibitor; PD, MEK-1/2 inhibitor; SB, p38 MAP kinase Immunoreactive bands were detected with horseradish inhibitor; SC, NF-jB inhibitor; SP, JNK-1/2 inhibitor. peroxidase-conjugated secondary antibody and developed using an enhanced chemiluminescence kit (Amersham SD. Differences between groups were assessed by independent Pharmacia Biotech, Piscataway, NJ, USA) and exposed to x- and paired t-tests. In all analyses, P < 0.05 was taken to ray film (Amersham Pharmacia Biotech). The immunoreac- indicate statistical significance. tive bands were quantified by densitometry and normalized relative to the b-actin level in the same sample. RESULTS Data Analysis miR-146a Expression in GO All experiments were performed at least three times indepen- dently, and using at least three cell cultures harvested from To determine which miRNAs are involved in the pathogenesis different individuals. The results are presented as means 6 of GO, we performed microarray analysis using GO (n ¼ 8) and

FIGURE 3. Effects of IL-1b on miR-146a expression in GO and non-GO orbital fibroblasts. Interleukin 1b induced a time- and concentration dependent increase in miR-146a expression in orbital fibroblasts from both GO and non-GO specimens (*P < 0.05, versus time and concentration- matched controls). We found that non-GO cells showed a similar response as GO orbital fibroblasts, but higher levels of miR-146a expression were observed in non-GO cells under some conditions (16 hours and 24 hours: 10 ng/mL; 16 hours: 1, 5, 10, and 20 ng/mL), compared with GO orbital fibroblasts (**P < 0.05, comparison between GO and healthy non-GO with IL-1b treatment). The results are expressed as the means 6 standard deviation of three individual samples and the graphs are representative of three independent experiments.

Downloaded from iovs.arvojournals.org on 09/28/2021 Role of miR-146a in Graves’ Orbitopathy IOVS j August 2016 j Vol. 57 j No. 10 j 4031

level at 6 hours (4.96 6 0.73-fold, P ¼ 0.019 compared with control, paired t test), which continued to rise to 17.05 6 1.38-fold at 16 hours and 18.63 6 1.41-fold at 24 hours (Fig. 3A). Interleukin 1b (16 hours) induced a concentration- dependent increase in miR-146a expression. Interleukin 1b caused an increase in miR-146a expression level by about 12- fold at 1 ng/mL (12.38 6 1.18-fold, P < 0.001 compared with the control, paired t-test), which continued to rise to 14.35 6 2.10-fold at 5 ng/mL, 17.54 6 2.70-fold at 10 ng/mL, and 18.75 6 2.42-fold at 20 ng/mL (Fig. 3B). Interleukin 1b induced a time- and concentration-dependent increase in miR-146a expression. The same experiments were performed using orbital fibroblasts from non-GO patients. The results showed a similar response to GO orbital fibroblasts, but higher levels of miR- 146a expression were observed in non-GO cells under some conditions (16 and 24 hours: 10 ng/mL; 16 hours: 1, 5, 10, and 20 ng/mL), compared with GO orbital fibroblasts (independent t-test, P < 0.05; Fig. 3).

Effects of Inhibitors of MEK-1/2, JNK-1/2, p38 MAP kinase, PI3-K and NF-jB on IL-1b-Induced miR-146a Expression To investigate the molecular mechanism underlying IL-1b– induced miR-146a expression, the effects of inhibitors of MEK- 1/2, JNK-1/2, p38 MAP kinase, and PI3-K were analyzed using FIGURE 5. Effects of miR-146a mimics and inhibitors on IL-1b–induced GO orbital fibroblasts. Interleukin 1b (10 ng/mL, 16 hours) IL-6 protein production expression in GO and non-GO orbital induced an increase of about 17.5-fold in miR-146 expression. fibroblasts. Interleukin 1b induced less IL-6 production by non-GO orbital fibroblasts (481.05 6 20.01 pg/mL) than GO orbital fibroblasts This increase in miR-146a expression induced by IL-1b was (526.59 6 19.55 pg/mL, **P ¼ 0.048). Interleukin 1b–induced IL-6 significantly inhibited by JNK-1/2 (5.28 6 0.34-fold, P ¼ 0.030 protein production was decreased by miR-146a mimics (30 and 100 compared with IL-1b–induced miR-146 expression, indepen- nM, *P < 0.05 compared with control mimics), whereas miR-146a dent t test) and PI3K inhibitors (9.73 6 2.32-fold, P ¼ 0.039), inhibitors did not induce any changes in IL-6 protein production in an but not by those of MEK-1/2 and p38 MAP kinase (Fig. 4). experiment using GO orbital fibroblasts. Interleukin 1b–induced IL-6 To assess the involvement of the NF-jB pathway, samples protein production was decreased by miR-146a mimics (10, 30 and 100 were pretreated with SC-514 and the miR-146a expression was nM, *P < 0.05 compared with control mimics), whereas miR-146a inhibitors did not induce any changes in IL-6 protein production in an examined. The SC-514 pretreatment significantly decreased the experiment using non GO orbital fibroblasts. The results are expressed level of miR-146a expression by (1.94 6 0.25-fold) compared as the means 6 standard deviation of three individual samples and the with the IL-1b–induced miR-146 expression (17.54 6 3.84- graphs are representative of three independent experiments. fold, P ¼ 0.019). Glucocorticoids have powerful anti-inflammatory actions. non-GO (n ¼ 6) orbital connective tissue. Microarray analyses Therefore, we examined whether dexamethasone affects the showed that 38 miRNAs were upregulated and 7 miRNAs were IL-1b–induced miR-146a expression. Dexamethasone pretreat- downregulated in orbital connective tissue from GO patients ment significantly decreased the level of miR-146a expression compared to healthy controls (Fig. 1). The heat map represents (4.95 6 0.48-fold) compared with the IL-1b–induced miR-146 the results using a color intensity scale with the highest and expression (17.54 6 3.84-fold, P ¼ 0.029). lowest expression levels corresponding to bright red and bright blue, respectively (Fig. 1A). Effects of miR-146a Mimics and Inhibitors on IL- Among the miRNAs overexpressed in GO, we noted that 1b–Induced IL-6 Protein Production miR-146a, which is a relatively well-known miRNA in inflammatory autoimmune diseases, was increased by 3.15- We transfected three GO and three non-GO orbital fibroblasts fold compared with normal controls. Six of the eight GO with 0, 10, 30, and 100 nM miR-146a mimics and inhibitors. In subjects had miR-146a expression higher than the mean an experiment using GO orbital fibroblasts, IL-1b induced IL-6 control value (Fig. 1). To confirm the results of microarray protein production (526.59 6 19.55 pg/mL) compared with analysis, we performed qPCR of miR-146a expression in RNA baseline (86.82 6 3.01 pg/mL). Interleukin 1b–induced IL-6 samples obtained from another five GO patients and five non- protein production was decreased by miR-146a mimics GO control subjects. The expression of miR-146a was (480.59 6 17.80 pg/mL at 30 nM and 418.15 6 15.83 pg/ significantly higher in orbital adipose tissues from GO than mL at 100 nM, P ¼ 0.017 and 0.001, respectively, compared from non-GO subjects (P ¼ 0.032, independent t-test; Fig. 2). with control mimics). Mimics of miR-146a induced a concen- tration-dependent decrease in IL-1b–induced IL-6 protein Effects of IL-1b on miR-146a Expression production, whereas miR-146a inhibitors did not induce any changes in IL-6 protein production (Fig. 5). Orbital fibroblasts from three GO and three non-GO patients The same experiments were performed using non-GO were used to measure miR-146a expression following exposure orbital fibroblasts. The results showed a similar response to to IL-1b. In an experiment using GO orbital fibroblasts, IL-1b GO orbital fibroblasts. Mimics of miR-146a induced a decrease (10 ng/mL) caused a 5-fold increase in miR-146a expression in the IL-1b–induced IL-6 protein production at concentrations

Downloaded from iovs.arvojournals.org on 09/28/2021 Role of miR-146a in Graves’ Orbitopathy IOVS j August 2016 j Vol. 57 j No. 10 j 4032

FIGURE 6. Effects of miR-146a mimics on IL-1b–induced IL-6, COX-2, and ICAM-1 protein production in GO orbital fibroblasts. The increases in IL-6 and ICAM-1 were inhibited by miR-146a mimics (30 nM) (*P < 0.05, versus IL-1b–induced IL-6 and ICAM-1 production). However, COX-2 did not show significant results. The results are expressed as the means 6 standard deviation of three individual samples and the graphs are representative of three independent experiments.

of 10, 30, and 100 nM, whereas miR-146a inhibitors did not al.18 reported strong miR-146a expression in cartilage in low- induce any change in the IL-6 protein production. grade OA. Sonkoly et al.15 reported that miR-146a was Of note, IL-1b induced less IL-6 production in non-GO significantly overexpressed in psoriatic lesional skin compared orbital fibroblasts (481.05 6 20.01 pg/mL) than in GO orbital with healthy skin. fibroblasts (526.59 6 19.55 pg/mL, P ¼ 0.048). In an Several recent pharmacologic studies investigated the experiment using non-GO orbital fibroblasts, IL-1b–induced mechanism of IL-1b–induced miR-146a expression using IL-6 protein production was significantly decreased by 10 nM different cell lines.26,27 Larner-Svensson et al.27 reported miR-146a mimics (413.39 6 15.34 pg/mL, P ¼ 0.011), whereas that IL-1b induced a time-dependent, 100-fold induction in 10 nM miR-146a mimics did not induce any changes in IL-6 miR-146a expression in primary human airway smooth protein production in GO orbital fibroblasts (Fig. 5). muscle cells. Perry et al.26 also reported that IL-1b induced a time- and concentration-dependent increase in miR-146a Effects of miR-146a Mimics on IL-1b–Induced IL-6, expression in human lung alveolar epithelial cells. In this COX-2, and ICAM-1 Protein Production study, we found that IL-1b induced time- and concentration- dependent increases in miR-146a expression in both GO and We further investigated the effects of miR-146a mimics on the non-GO orbital fibroblasts. Interestingly, the expression of expression of inflammatory proteins, such as IL-6, COX-2, and miR-146a was upregulated to a lesser extent in GO orbital ICAM-1, by Western blotting using orbital fibroblasts from GO. fibroblasts than in non-GO orbital fibroblasts after stimula- Stimulation with IL-1b increased the levels of IL-6, COX-2, and tion with IL-1b (P < 0.05). This result is in line with studies ICAM-1 expression. The increases in IL-6 and ICAM-1 were that showed that GO orbital fibroblasts produced signifi- inhibited by miR-146a mimics (30 nM), but no significant effect cantly lower levels of IL-1 receptor antagonist (IL-1RA) was observed on COX-2 expression (Fig. 6). compared with non-GO orbital fibroblasts.28 Because IL-1RA acts as a competitive inhibitor of IL-1a and IL-1b and reduces the inflammatory process in GO orbital tissues,28–30 we DISCUSSION believe that these findings support the hypothesis that GO This study investigated the role of miR-146a in regulation of orbital fibroblasts have a reduced capacity to control the inflammation in an in vitro model of GO. We compared the inflammatory response. expression level of miR-146a between GO and non-GO orbital Inhibitors of MEK-1/2, JNK-1/2, p38 MAP kinase, and PI3-K adipose tissue. The expression of miR-146a was significantly were used to investigate which signal cascade controls the higher in orbital adipose tissues from GO than from non-GO increase in miR-146a expression by IL-1b. The results indicated subjects (P ¼ 0.032). GO is an inflammatory autoimmune that the increase in miR-146a expression induced by IL-1b was disease, and thus miR-146a seems to be upregulated by inhibited by JNK-1/2 and PI3K inhibitors, suggesting that inflammatory stress, as in GO. Similarly, there have been activation of JNK and PI3K pathways was required for IL-1b– several reports that expression of miR-146a was increased in induced miR-146a expression in orbital fibroblasts. Larner- diseased tissue.11,15,17,18 Nakasa et al.11 reported that miR-146a Svensson et al.27 reported similar experimental results was highly expressed in RA synovial tissue, and Yamasaki et indicating that IL-1b–induced miR-146a was regulated by

Downloaded from iovs.arvojournals.org on 09/28/2021 Role of miR-146a in Graves’ Orbitopathy IOVS j August 2016 j Vol. 57 j No. 10 j 4033

MEK-1/2 and JNK-1/2 in human airway smooth muscle cells. In Acknowledgments their report, the extent of the decrease in miR-146a expression Supported by a grant from the National Research Foundation of was greater for JNK-1/2 than MEK-1/2. In the present study, the Korea (2014R1A1002754) and in part by the Soonchunhyang JNK-1/2 pathway, but not MEK1/2, was associated with IL-1b– University Research Fund. induced upregulation of miR146a. As the function and mechanism of action of miR-146a are dependent on the cell Disclosure: S.Y. Jang, None; M.K. Chae, None; J.H. Lee, None; type,27 we assumed that this discrepancy was due to the E.J. Lee, None; J.S. Yoon, None different cell lines used in these studies. The observation that the PI3K pathway was required for IL-1b–induced miR-146a References expression in orbital fibroblasts can be explained. Recently, a cAMP-independent cascade was shown to increase PI3K 1. Grubeck-Loebenstein B, Trieb K, Sztankay A, Holter W, Anderl activity, a signaling pathway that plays a central role in the H, Wick G. Retrobulbar T cells from patients with Graves’ pathogenesis of GO.31 Kumar et al.31 reported that a ophthalmopathy are CD8þ and specifically recognize autolo- stimulatory TSH receptor antibody enhanced adipogenesis via gous fibroblasts. J Clin Invest. 1994;93:2738–2743. PI3K activation in GO, and suggested that inhibition of PI3K 2. Otto EA, Ochs K, Hansen C, Wall JR, Kahaly GJ. Orbital tissue- signaling may represent a potential novel therapeutic approach derived T lymphocytes from patients with Graves’ ophthal- in GO. mopathy recognize autologous orbital antigens. JClin Dexamethasone, a corticosteroid, attenuates the actions of Endocrinol Metab. 1996;81:3045–3050. multiple proinflammatory transcription factors, including NF- 3. Feldon SE, Park DJ, O’Loughlin CW, et al. Autologous T- jB. We found that IL-1b–induced miR146a expression was lymphocytes stimulate proliferation of orbital fibroblasts inhibited after treatment with dexamethasone. Moreover, we derived from patients with Graves’ ophthalmopathy. Invest found that the level of miR-146a expression was significantly Ophthalmol Vis Sci. 2005;46:3913–3921. decreased by SC-514, which is a selective NF-jB inhibitor. 4. Pappa A, Lawson JM, Calder V, Fells P, Lightman S. T cells and According to a previous report,20 miR-146a acts through fibroblasts in affected extraocular muscles in early and late inhibition of the NF-jB pathway by downregulating its target thyroid associated ophthalmopathy. Br J Ophthalmol. 2000; genes, such as TRAF6 and IRAK1. This leads to the termination 84:517–522. or mitigation of the inflammatory response. 5. Kuriyan AE, Phipps RP, Feldon SE. The eye and thyroid disease. In this study, we found that IL-1b–induced IL-6 protein Curr Opin Ophthalmol. 2008;19:499–506. production was further decreased by miR-146a mimics, 6. Bahn RS. Clinical review 157: Pathophysiology of Graves’ confirming the positive effect of miR-146a on the anti- ophthalmopathy: the cycle of disease. J Clin Endocrinol inflammatory process. We further investigated whether miR- Metab. 2003;88:1939–1946. 146a mimics could affect the levels of other inflammatory 7. Iyer S, Bahn R. Immunopathogenesis of Graves’ ophthalmop- cytokines induced by IL-1b and found that increases in IL-6 athy: the role of the TSH receptor. Best Pract Res Clin and ICAM-1 expression by IL-1b were inhibited by miR-146a Endocrinol Metab. 2012;26:281–289. mimics (30 nM). The increase in COX-2 expression by IL-1b 8. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and was decreased by miR-146a mimics, although the effect was function. Cell. 2004;116:281–297. 32 not statistically significantly. Recently, Wei et al. reported 9. Bartel DP. MicroRNAs: target recognition and regulatory significantly lower circulating serum levels of miR-146a in functions. Cell. 2009;136:215–233. GO patients compared with controls, and suggested that 10. Sonkoly E, Pivarcsi A. MicroRNAs in inflammation. Int Rev weakened miR-146a activity caused an increase in inflam- Immunol. 2009;28:535–561. mation. The authors further showed that the serum levels of miR-146a were significantly correlated with clinical activity 11. Nakasa T, Miyaki S, Okubo A, et al. Expression of microRNA- score, which indicates the disease inflammatory activity of 146 in rheumatoid arthritis synovial tissue. Arthritis Rheum. 2008;58:1284–1292. GO. Attenuation of miR-146a activity using an miR-146a 12. Dai Y, Huang YS, Tang M, et al. Microarray analysis of inhibitor had no significant effect on IL-1b–induced IL-6 microRNA expression in peripheral blood cells of systemic release in this study. According to a previous review,33 there lupus erythematosus patients. Lupus. 2007;16:939–946. are multiple and complex pathways for the release of IL-6 by 13. Tang Y, Luo X, Cui H, et al. MicroRNA-146A contributes to cells, and cells use different pathways, organelles, carriers, abnormal activation of the type I interferon pathway in human and molecules to control the release of cytokines. As the lupus by targeting the key signaling proteins. Arthritis molecular mechanism of IL-1b–induced IL-6 release in orbital Rheum. 2009;60:1065–1075. fibroblasts has not yet been fully elucidated, further 14. Wu F, Zikusoka M, Trindade A, et al. MicroRNAs are physiological studies are needed to determine why miR- differentially expressed in ulcerative colitis and alter expres- 146a inhibitors did not induce any changes in IL-1b–induced sion of macrophage inflammatory peptide-2 alpha. Gastroen- IL-6 release. Although several questions could not be terology. 2008;135:1624–1635.e1624. answered by the results of this study, we showed for the 15. Sonkoly E, Wei T, Janson PC, et al. MicroRNAs: novel first time that miR-146a may play a role in the regulation of regulators involved in the pathogenesis of psoriasis? PLoS inflammation in orbital fibroblasts from GO and that it One. 2007;2:e610. participates in the pathogenesis of GO. 16. Xu WD, Lu MM, Pan HF, Ye DQ. Association of MicroRNA-146a In conclusion, miR-146a seems to contribute to GO with autoimmune diseases. Inflammation. 2012;35:1525– pathogenesis by modulating inflammatory protein expression 1529. and cellular functions in orbital fibroblasts. MicroRNA 146a 17. Stanczyk J, Pedrioli DM, Brentano F, et al. Altered expression of was upregulated by inflammatory stress, such as IL-1b, and our microRNA in synovial fibroblasts and synovial tissue in results indicated a positive effect of miR-146a on the anti- rheumatoid arthritis. Arthritis Rheum. 2008;58:1001–1009. inflammatory process. Further studies are required to examine 18. Yamasaki K, Nakasa T, Miyaki S, et al. Expression of microRNA- the potential of miR-146a as a target for therapeutic strategies 146a in osteoarthritis cartilage. Arthritis Rheum. 2009;60: and as a biomarker. 1035–1041.

Downloaded from iovs.arvojournals.org on 09/28/2021 Role of miR-146a in Graves’ Orbitopathy IOVS j August 2016 j Vol. 57 j No. 10 j 4034

19. Alevizos I, Illei GG. MicroRNAs in Sjogren’s syndrome as a 27. Larner-Svensson HM, Williams AE, Tsitsiou E, et al. Pharmaco- prototypic autoimmune disease. Autoimmun Rev. 2010;9: logical studies of the mechanism and function of interleukin- 618–621. 1beta-induced miRNA-146a expression in primary human 20. Taganov KD, Boldin MP, Chang KJ, Baltimore D. NF-kappaB- airway smooth muscle. Respir Res. 2010;11:68. dependent induction of microRNA miR-146, an inhibitor 28. Muhlberg T, Heberling HJ, Joba W, Schworm HD, Heufelder A. targeted to signaling proteins of innate immune responses. Detection and modulation of interleukin-1 receptor antagonist Proc Natl Acad Sci USA. 2006;103:12481–12486. messenger ribonucleic acid and immunoreactivity in Graves’ 21. Yoon JS, Chae MK, Jang SY, Lee SY, Lee EJ. Antifibrotic effects orbital fibroblasts. Invest Ophthalmol Vis Sci. 1997;38:1018– of quercetin in primary orbital fibroblasts and orbital fat tissue 1028. cultures of Graves’ orbitopathy. Invest Ophthalmol Vis Sci. 29. Muhlberg T, Joba W, Spitzweg C, Schworm HD, Heberling HJ, 2012;53:5921–5929. Heufelder AE. Interleukin-1 receptor antagonist ribonucleic 22. Yoon JS, Lee HJ, Chae MK, Lee SY, Lee EJ. Cigarette smoke acid and protein expression by cultured Graves’ and normal extract–induced adipogenesis in Graves’ orbital fibroblasts is orbital fibroblasts is differentially modulated by dexametha- inhibited by quercetin via reduction in oxidative stress. J sone and irradiation. J Clin Endocrinol Metab. 2000;85:734– Endocrinol. 2013;216:145–156. 742. 23. Rhiu S, Chae MK, Lee EJ, Lee JB, Yoon JS. Effect of tanshinone 30. Smith TJ. Orbital fibroblasts exhibit a novel pattern of IIA in an in vitro model of Graves’ orbitopathy. Invest responses to proinflammatory cytokines: potential basis for Ophthalmol Vis Sci. 2014;55:5900–5910. the pathogenesis of thyroid-associated ophthalmopathy. Thy- 24. Livak KJ, Schmittgen TD. Analysis of relative gene expression roid. 2002;12:197–203. data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–408. 31. Kumar S, Nadeem S, Stan MN, Coenen M, Bahn RS. A stimulatory TSH receptor antibody enhances adipogenesis 25. Yoon JS, Lee HJ, Choi SH, Chang EJ, Lee SY, Lee EJ. Quercetin via phosphoinositide 3-kinase activation in orbital preadipo- inhibits IL-1beta-induced inflammation, hyaluronan produc- cytes from patients with Graves’ ophthalmopathy. J Mol tion and adipogenesis in orbital fibroblasts from Graves’ Endocrinol. 2011;46:155–163. orbitopathy. PLoS One. 2011;6:e26261. 26. Perry MM, Moschos SA, Williams AE, Shepherd NJ, Larner- 32. Wei H, Guan M, Qin Y, et al. Circulating levels of miR-146a and Svensson HM, Lindsay MA. Rapid changes in microRNA-146a IL-17 are significantly correlated with the clinical activity of expression negatively regulate the IL-1beta-induced inflamma- Graves’ ophthalmopathy. Endocr J. 2014;61:1087–1092. tory response in human lung alveolar epithelial cells. J 33. Stanley AC, Lacy P. Pathways for cytokine secretion. Physiol- Immunol. 2008;180:5689–5698. ogy (Bethesda). 2010;25:218–229.

Downloaded from iovs.arvojournals.org on 09/28/2021