CXCL9 and CXCL11 Chemokines Modulation by Peroxisome ...

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CXCL9 and CXCL11 Chemokines Modulation by Peroxisome Proliferator-Activated Receptor-␣ Agonists Secretion in Graves’ and Normal Thyrocytes Alessandro Antonelli, Silvia Martina Ferrari, Silvia Frascerra, Cinzia Pupilli, Caterina Mancusi, Maria Rita Metelli, Claudio Orlando, Ele Ferrannini, and Poupak Fallahi Department of Internal Medicine (A.A., S.M.F., S.F., C.M., E.F., P.F.), University of Pisa, School of Medicine, I-56100 Pisa, Italy; Endocrinology Unit (C.P.), Azienda Ospedaliera Careggi and University of Florence, 50139 Florence, Italy; Medicina di Laboratorio e Diagnostica Molecolare (M.R.M.), Azienda Ospedaliera Pisana, 56126 Pisa, Italy; and Department of Clinical Pathophysiology (C.O.), Clinical Biochemistry Unit, University of Florence, 50139 Florence; Italy Context: Peroxisome proliferator-activated receptor (PPAR)-␣ has been shown to exert immunomodulatory effects in autoimmune disorders. However, until now, no data were present in the literature about the effect of PPAR␣ activation on CXCL9 and CXCL11 chemokines in general or on secretion of these chemokines in thyroid cells. Objective and Design: The presence of PPAR␣ and PPAR␥ has been evaluated by real-time-PCR in Graves’ disease (GD) and control cells in primary culture. Furthermore, we have tested the role of PPAR␣ and PPAR␥ activation on CXCL9 and CXCL11 secretion in GD and control cells after stimulation of these chemokines secretion with IFN␥ and TNF␣. Results: This study shows the presence of PPAR␣ and PPAR␥ in GD and control cells. A potent dose-dependent inhibition by PPAR␣-agonists was observed on the cytokines-stimulated secretion of CXCL9 and CXCL11 in GD and control cells. The potency of the PPAR␣ agonists used was maximum on the secretion of CXCL9, reaching about 90% of inhibition by fenofibrate and 85% by ciprofibrate. The relative potency of the compounds was different with each chemokine; for example, gemfibrozil exerted a 55% inhibition on CXCL11, whereas it had a weaker activity on CXCL9 (40% inhibition). PPAR␣ agonists were stronger (ANOVA, P ⬍ 0.001) inhibitors of CXCL9 and CXCL11 secretion in thyrocytes than PPAR␥ agonists. Conclusions: Our study shows the presence of PPAR␣ in GD and control thyrocytes. PPAR␣ activators are potent inhibitors of the secretion of CXCL9 and CXCL11, suggesting that PPAR␣ may be involved in the modulation of the immune response in the thyroid. (J Clin Endocrinol Metab 95: E413–E420, 2010)

he peroxisome proliferator-activated receptors (PPAR␣, ␥, and ␦) are ligand-activated nuclear receptors with a wide range of effects on metabolism, cellular proliferation, differentiation, and immune response (1–3). Of considerable interest, ligands for PPAR␥ and PPAR␣ have therapeutic activity in several rodent models of inflammatory and autoimmune disease (4 –14), sug-

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gesting that they might have similar activity in human disease as well. The PPARs are expressed in dendritic cells, macrophages, B and T lymphocytes, and endothelial and epithelial cells. Thus, several cell types are potential targets for the antiinflammatory effects of PPAR ligands (4 –14). PPAR␣ ligands have also been shown to regulate inflamma-

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2010 by The Endocrine Society doi: 10.1210/jc.2010-0923 Received April 22, 2010. Accepted August 3, 2010. First Published Online September 1, 2010

Abbreviations: GD, Graves’ disease; IFN, interferon; NF-␬B, nuclear factor-␬B; PPAR, peroxisome proliferator-activated receptor; RGZ, rosiglitazone; RT, real-time.

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tory responses; in fact, they are expressed on T cells, and their ligands can inhibit IL-2 production and T-cell proliferation (15). PPAR␣ ligands inhibit IL-6, vascular cell adhesion molecule, and cyclooxygenase-2 activated by cytokines (16). PPAR␣ ligands may inhibit the activity of nuclear factor-␬B (NF-␬B), in part by increasing the expression of inhibitor of ␬B (17). Recently, it has been shown that fenofibrate represses IL-17 and interferon (IFN)-␥ expression and improves colitis in IL-10-deficient mice by inhibiting the expression of the gene encoding chemokine (C-X-C motif) ligand 10 (CXCL10) and by repressing CXCL10 gene promoter activity in TNF-␣-treated human colon adenocarcinoma grade II (HT-29) cells (7). Recent evidence has shown that CXC ␣-chemokines (Th1), especially CXCL9, CXCL10, and CXCL11, play an important role in the initial phases of autoimmune thyroid disorders (18 –22). Serum CXCL10 levels are increased in Graves’ disease (GD), especially in patients with active disease, and the CXCL10 decrease after thyroidectomy (23) or after radioiodine (24) shows that it is more likely to have been produced inside the thyroid gland. Furthermore, patients with newly diagnosed autoimmune thyroiditis show increased serum CXCL10, overall in the presence of a more aggressive thyroiditis and hypothyroidism (19, 20). We have recently shown that the secretion of CXCL9 and CXCL11 in primary cultures of GD thyrocytes can be stimulated by IFN␥ and TNF-␣ (25, 26), suggesting that GD thyrocytes participate in the self-perpetuation of inflammation by releasing chemokines (under the influence of cytokines) and inducing the recruitment of activated T cells in the thyroid. Treatment of thyroid follicular cells with PPAR␥ activators, at near-therapeutic doses, significantly inhibited IFN␥-stimulated CXC chemokine secretion, strongly suggesting that PPAR␥ might be involved in the regulation of IFN␥-induced chemokine expression in human thyroid autoimmunity and GD (25, 26). Recently, PPAR␣ has been shown to be expressed in thyroid cells (27). However, until now, to our knowledge, no data are present in literature about the effect of PPAR␣ activation on chemokines secretion in thyroid cells or on CXCL9 or CXCL11 in general. Here, we have tested the possible modulatory role of PPAR␣ activation on the CXC chemokines CXCL9 and CXCL11 secretion in GD and control thyrocytes in primary culture.

thy). GD patients were advised to have thyroidectomy (after a previous methimazole course in the presence of a large goiter and/or thyroid nodules), mainly due to a relapse of hyperthyroidism. In addition, control samples of normal thyroid tissue were collected from five patients (two undergoing parathyroidectomy, three laryngeal intervention) (mean age, 49 yr; range, 32– 64; three females, two males). All study subjects gave their informed consent to the study, which was approved by the local ethical committee. Thyrocytes were prepared as reported previously (21, 25, 26). The tissue samples were digested by collagenase (1 mg/ml; Roche, Mannheim, Germany) in RPMI 1640 (Whittaker Bioproducts, Inc., Walkersville, MD) for 1 h at 37 C. Removed semidigested follicles were sedimented for 2 min, washed, and cultured in RPMI 1640 medium containing 10% fetal bovine serum (Seromed; Biochrom, Berlin, Germany), 2 mM glutamine, and 50 ␮g/ml penicillin/streptomycin at 37 C and 5% CO2.

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Patients and Methods Thyroid follicular cells Samples of thyroid tissue were obtained from five euthyroid patients with GD undergoing surgery (mean age, 43 yr; range, 26 –57; four females, one male; without Graves’ ophthalmopa-

CXCL9 and CXCL11 secretion assays For CXCL9 and CXCL11 secretion assays, cells were seeded in 96-well plates at a concentration of 30,000 cells/ml in a final volume of 100 ␮l per well in growth medium, which was removed after 24 h. Then, cells were washed in PBS and incubated (24 h) in phenol red and serum-free medium with IFN␥ (R&D Systems, Minneapolis, MN; 500, 1,000, 5,000, and 10,000 IU/ ml) and 10 ng/ml TNF-␣ (R&D Systems), alone or in combination (25). The concentration of TNF-␣ was selected in preliminary experiments to obtain the highest responses. After 24 h, the supernatant was collected and kept frozen at ⫺20 C until CXCL10 assay. To establish how the PPAR␣ activators affect the IFN␥-induced chemokine secretion, cells were treated (24 h) with IFN␥ (1000 IU/ml) and TNF-␣ (10 ng/ml) in the presence or absence of increasing concentrations of pure PPAR␣ agonists (5, 10, 50, and 100 ␮M fenofibrate; 50, 100, 200, and 400 ␮M gemfibrozil or ciprofibrate) (Sigma-Aldrich Corp., St. Louis, MO). To establish how the PPAR␥ activators affect the IFN␥-induced chemokine secretion, cells were treated (24 h) with IFN␥ (1000 IU/ml) and TNF-␣ (10 ng/ml) in the presence or absence of increasing concentrations (0, 1, 5, 10, and 20 ␮M) of the pure PPAR␥ agonist, rosiglitazone (RGZ; Glaxo, Welwyn, UK), or pioglitazone (Alexis Biochemicals, Lausen, Switzerland). Supernatants were assayed by ELISA for CXCL9 and CXCL11 concentrations. The experiments were repeated three times with the three different cell preparations.

Cell cultures and PPAR␣ or PPAR␥ agonist treatment Cultures of thyrocytes were treated (24 h) with 0, 1, 5, 10, or 20 ␮M RGZ or pioglitazone, or PPAR␣ agonists (5, 10, 50, 100 ␮M fenofibrate; 50, 100, 200, 400 ␮M gemfibrozil or ciprofibrate). Control cultures were grown (24 h) in the same medium containing vehicle (absolute ethanol, 0.47% vol/vol) without PPAR␣ or PPAR␥ agonists. Some cultures were examined by phase contrast microscopy using an Olympus IX50. Lysis and homogenization of cell preparations were performed, and the sample was immediately assayed for its protein concentration by conventional methods (25).

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ELISA for CXCL9 and CXCL11 CXCL9 and CXCL11 levels were measured in culture supernatants by commercially available kits (R&D Systems). The mean minimum detectable dose was 1.37 pg/ml for CXCL9 and 3.4 pg/ml for CXCL11; the intra- and interassay coefficients of variation were 3.3 and 6.2% for CXCL9, and 4.7 and 7.6% for CXCL11. Quality control pools of low, normal, or high concentration for all parameters were included in each assay.

Real-time (RT)-PCR for PPAR␣ and PPAR␥ Total RNA from the cells was extracted with the RNeasy Mini reagent kit (QIAGEN, Milan, Italy) according to the manufacturer’s recommendations. TaqMan Reverse Transcription Reagents kit and Universal PCR Master Mix were from Applied Biosystems (Foster City, CA). Quantitative PCR human reference total RNA was purchased from Stratagene (La Jolla, CA). Primers and probes for PPAR␣ and PPAR␥ were from Applied Biosystems (TaqMan Gene Expression Assay; Hs00231882_m1 and Hs00234592_m1, respectively). Total RNA (400 ng) was reverse-transcribed using TaqMan Reverse Transcription Reagents kit. Reverse transcription was performed in a final volume of 80 ml containing 500 mM KCl, 0.1 mM EDTA, 100 mM Tris-HCl (pH 8.3), 5.5 mM MgCl2, 500 ␮M each of deoxyribonucleotide triphosphate, 2.5 ␮M random examers, 0.4 IU/␮l RNase inhibitor, and 1.25 IU/␮l Multiscribe Reverse

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Transcriptase. The reverse transcription reaction was performed at 25 C for 10 min, 48 C for 30 min, and 95 C for 3 min. Measurement of gene expression was performed by quantitative RT-PCR (TaqMan). For each sample, 12.5 ng of cDNA was added to 10 ␮l of PCR mix containing each primer set and 1⫻ Universal PCR Master Mix. The samples were then subjected to 40 cycles of amplification at 95 C for 15 sec and 60 C for 60 sec in the ABI Prism 7700 Sequence Detector (Applied Biosystems). The amount of target, normalized to the endogenous reference glyceraldehyde 3-phosphate dehydrogenase (Pre-Developed TaqMan Assay Reagents, Applied Biosystems) and relative to a calibrator (Quantitative PCR human reference total RNA), was given by 2⫺⌬⌬Ct calculation (28).

Data analysis Values are given as mean ⫾ SD for normally distributed variables (in text), or mean ⫾ SEM (in figures), otherwise as median [interquartile range]. Mean group values were compared by using oneway ANOVA for normally distributed variables, otherwise by the Mann-Whitney U or Kruskal-Wallis test. Proportions were compared by the ␹2 test. Post hoc comparisons on normally distributed variables were carried out using the Bonferroni-Dunn test.

Results

FIG. 1. Increasing doses of fenofibrate (5, 10, 50, 100 ␮M) inhibit CXCL9 or CXCL11 release from thyrocytes obtained from patients with GD (A and C, respectively) or control thyroid follicular cells (B and D, respectively), stimulated with IFN␥ (1000 IU/ml) and TNF-␣ (10 ng/ml) (IFNg⫹TNFa) (P ⬍ 0.0001 by ANOVA). Bars represent mean ⫾ SEM. *, P ⬍ 0.05 or less vs. IFNg⫹TNFa by Bonferroni-Dunn test. Feno, Fenofibrate.

PPAR␣ and PPAR␥ mRNAs were detectable in all primary thyrocytes. According to RT-PCR results, the PPAR␣ expression vs. the reference gene (GAPDH) ranges from 0.33 to 0.57 in GD thyrocytes and from 0.21 to 0.47 in control thyrocytes. Moreover, the expression of PPAR␥ ranges from 0.04 to 0.66 in GD thyrocytes and from 0.06 to 0.52 in control thyrocytes. CXCL9 was undetectable in the supernatants collected from primary GD thyrocyte cultures. IFN␥ dose-dependently induced the CXCL9 release (CXCL9, 0, 65 ⫾ 21, 143 ⫾ 29, 201 ⫾ 37, and 246 ⫾ 43 pg/ml, respectively; with IFN␥, 0, 500, 1,000, 5,000, 10,000 IU/ml; ANOVA, P ⬍ 0.001). TNF-␣ alone had no effect (chemokines remaining undetectable), whereas the combination of IFN␥ and TNF-␣ had a significant synergistic effect on the CXCL9 secretion (CXCL9, 10,273 ⫾ 1,394 vs. 145 ⫾ 27 pg/ml with IFN␥ alone; ANOVA, P ⬍ 0.0001). Treating thyrocytes with RGZ or pioglitazone together with IFN␥ and TNF-␣ stimulation, CXCL9 release was dosedependently inhibited (ANOVA, P ⬍ 0.0001), as expected (25, 26).

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effect on the CXCL11 secretion (CXCL11, 1894 ⫾ 126 vs. 98 ⫾ 16 pg/ml with IFN␥ alone; ANOVA, P ⬍ 0.0001). Treating thyrocytes with RGZ or pioglitazone together with IFN␥ and TNF-␣ stimulation, CXCL11 release was dosedependently inhibited (ANOVA, P ⬍ 0.0001), as expected (25, 26). Treating GD thyrocytes with fenofibrate (Fig. 1C), or ciprofibrate (Fig. 2C) or gemfibrozil (Fig. 3C), together with IFN␥ and TNF-␣ stimulation, CXCL11 release was dose-dependently inhibited. The inhibition of the CXCL11 chemokine secretion induced by PPAR␣ agonists was stronger than that induced by PPAR␥ activators in GD thyrocytes (Fig. 4B). However, the relative potency of the PPAR␣ agonists on the inhibition was different for CXCL11 chemokine, with the highest inhibitory effect obtained with fenofibrate (80% of inhibition), with respect to ciprofibrate (57% of inhibition) or gemfibrozil (55% of inhibition). Interestingly, the relative potency of each compound was different with each chemokine; gemfibrozil, FIG. 2. Increasing doses of ciprofibrate (50, 100, 200, 400 ␮M) inhibit CXCL9 or CXCL11 for example, was able to inhibit CXCL11 release from thyrocytes obtained from patients with GD (A and C, respectively) or control thyroid follicular cells (B and D, respectively), stimulated with IFN␥ (1000 IU/ml) and TNF-␣ (10 secretion by 55%, but it had a weaker ng/ml) (IFNg⫹TNFa) (P ⬍ 0.0001 by ANOVA). Bars represent mean ⫾ SEM. *, P ⬍ 0.05 or less effect on CXCL9 secretion (about a vs. IFNg⫹TNFa by Bonferroni-Dunn test. Cipro, Ciprofibrate. 40% inhibition). RGZ or pioglitazone, fenofibrate, ciprofibrate, or gemfibrozil alone had Treating GD thyrocytes with fenofibrate (Fig. 1A), ciprofibrate (Fig. 2A), or gemfibrozil (Fig. 3A), together with no effect and did not affect cell viability or total protein IFN␥ and TNF-␣ stimulation, CXCL9 release was dose- content (data not shown). The data obtained with thyrocytes from normal thyroid tissue (Figs. 1, B–D; 2, B–D; and dependently inhibited. The inhibition of the CXCL9 chemokine secretion in- 3, B–D) were not statistically different from those obduced by PPAR␣ agonists was stronger than that induced tained from GD (data not shown). by PPAR␥ activators in GD thyrocytes (Fig. 4A). However, the relative potency of the PPAR␣ agonists on the inhibition was different on CXCL9 release; in fact, gemfibrozil has a weaker inhibitory effect (40% of inhibition) in comparison with ciprofibrate (85% of inhibition) and fenofibrate (90% of inhibition). Also, CXCL11 was undetectable in the supernatants collected from primary GD thyrocyte cultures. IFN␥ dosedependently induced the CXCL11 release (CXCL11, 0, 41 ⫾ 16, 102 ⫾ 24, 192 ⫾ 35, and 211 ⫾ 41 pg/ml, respectively; with IFN␥, 0, 500, 1,000, 5,000, and 10,000 IU/ml; ANOVA, P ⬍ 0.001). TNF-␣ alone had no effect (chemokines remaining undetectable), whereas the combination of IFN␥ and TNF-␣ had a significant synergistic

Discussion Our study confirms that IFN␥ and TNF-␣ stimulated CXC chemokine CXCL9 and CXCL11 secretion as expected (25, 26). These results, on the whole, are in agreement with the view that autoimmune disorders evolve from an initial Th1 phase (18). In fact, IFN␥-inducible CXC chemokines can be produced by several types of normal mammalian cells, such as thyrocytes, fibroblasts, colon epithelial cells, islet cells, and others (18 –26). However, these cells are not able to produce the CXC chemokines in basal condition, but only after the stimulation by cytokines, such as IFN␥ and

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vocate PPAR␣ agonists as therapy for autoimmune disease (5). In fact, the PPAR agonists, gemfibrozil, ciprofibrate, and fenofibrate, have been shown to increase the production of the Th2 cytokine, IL-4, and suppress proliferation by T-cell receptor transgenic T cells specific for the myelin basic protein Ac111. Oral administration of gemfibrozil and fenofibrate inhibited clinical signs of experimental autoimmune encephalomyelitis. More importantly, gemfibrozil was shown to shift the cytokine secretion of human T-cell lines by inhibiting IFN␥ and promoting IL-4 secretion. These results have suggested that PPAR␣ agonists, such as gemfibrozil and fenofibrate, may be attractive candidates for use in human inflammatory conditions such as multiple sclerosis (5). Additionally, PPAR␣ agonists inhibited the secretion of the proinflammatory cytokines TNF-␣, IL-1␤, and IL-6 by lipopolysaccharide-stimulated astrocytes (29). The effect of PPAR␣ on CXC chemokines has been less deeply investiFIG. 3. Increasing doses of gemfibrozil (50, 100, 200, 400 ␮M) inhibit CXCL9 or CXCL11 gated. In the study by Lee et al. (7), release from thyrocytes obtained from patients with GD (A and C, respectively) or control thyroid follicular cells (B and D, respectively), stimulated with IFN␥ (1000 IU/ml) and TNF-␣ (10 treatment of C3H.IL-10 (⫺/⫺) mice ng/ml) (IFNg⫹TNFa) (P ⬍ 0.0001 by ANOVA). Bars represent mean ⫾ SEM. *, P ⬍ 0.05 or less with fenofibrate delayed the onset of vs. IFNg⫹TNFa by Bonferroni-Dunn test. Gem, Gemfibrozil. colitis, decreased the colonic histopathology score, and decreased colonic TNF-␣, which are secreted in a Th1-type inflammatory expression of genes encoding the inflammatory cytokines site, such as the thyroid at the beginning of GD, by TIFN␥ and IL-17. The target for fenofibrate, PPAR␣, was helper 1-activated lymphocytes. This process has been expressed in lymphocytes, macrophages, crypt and sursuggested to be involved in the initiation and the perpetface epithelial cells of the colon. Fenofibrate repressed the uation of the inflammation in several autoimmune disexpression of the gene encoding CXCL10, and repressed eases (18 –26) and, on the basis of our results, can be apCXCL10 gene promoter activity in TNF-␣-treated HT-29 plied to the thyroid, too, in GD. cells. The authors conclude that the novel therapeutic acPPAR␣ has been shown to be expressed in both GD and normal thyroid cells. This result confirms results obtained tivity of fenofibrate in this mouse model suggests that it by Kasai et al. (27) using cells prepared from two patients may also have activity in Crohn’s disease. To the best of our knowledge, no study has investiwith GD. Moreover, in the present study, we show that PPAR␣ are expressed in normal thyroid cells in culture, at gated the PPAR␣ effect on CXCL9 and CXCL11 chemokines. The results of this study shed new light on the a level similar to that observed in GD. The functional meaning of PPAR␣ in thyroid cells has PPAR␣ effect on CXC chemokines, demonstrating a not been investigated until now. Our results suggest that powerful effect on the secretion of both CXCL9 and PPAR␣ can modulate the Th1 immune response, as sug- CXCL11 in normal thyrocytes, such as in cells obtained gested, by their powerful action on the inhibition of che- from GD, inhibiting the stimulatory effect of IFN␥ and mokines. In fact, PPAR␣ are able to modulate both TNF-␣ on the secretion of these chemokines. Interestingly, the effect of PPAR␣ has been exerted at nearCXCL9 and CXCL11 chemokines. The antiinflammatory action of PPAR␣ has been therapeutic doses, as suggested also by the study of widely investigated by many studies, and some studies ad- Lovett-Racke et al. (5).

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FIG. 4. The inhibition of the CXCL9 (A) or CXCL11 (B) chemokine secretion induced by PPAR␣ agonists in GD thyrocytes at the maximum dosage [fenofibrate (FENO), 100 ␮M; gemfibrozil (GEMF), 400 ␮M; ciprofibrate (CIPRO), 400 ␮M兴 was stronger than that induced by PPAR␥ activators [pioglitazone (PIO), 20 ␮M; rosiglitazone (ROSI), 20 ␮M]. Gemfibrozil has a weaker inhibitory effect on CXCL9 secretion in comparison with ciprofibrate and fenofibrate. The highest inhibitory effect on CXCL11 was obtained with fenofibrate. Bars represent mean ⫾ SEM. *, P ⬍ 0.05 or less vs. IFN␥⫹TNF-␣ (IFN⫹TNF) by Bonferroni-Dunn test; o, P ⬍ 0.05 or less vs. IFN⫹TNF⫹CIPRO400 or IFN⫹TNF⫹FENO100 or IFN⫹TNF⫹GEMF400.

The relative potency of each compound was different with each chemokine. This may suggest that PPAR␣ agonists act through different pathways that remain to be explored. In the study by Lee et al. (7), fenofibrate repressed the expression of a CXCL10 promoter-luciferase construct, indicating that the repression of CXCL10 mRNA expression occurs at least partly by a transcriptional mechanism. TNF-␣-stimulated transcription of the CXCL10 gene is largely mediated by NF-␬B binding to two ␬B binding sites located in the CXCL10 proximal promoter (30).

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PPAR ligands repress transcriptional activation by NF-␬B via a mechanism known as ligand-dependent transrepression (16, 31, 32); thus, NF-␬B is a likely target for repression of CXCL10 transcription by fenofibrate. Interestingly, however, in the study by Lee et al. (7), all five tested chemokine genes are known to be activated by NF-␬B (33–36), but two of these genes, CXCL8(IL-8) and CXCL1(GRO), were not repressed significantly by fenofibrate. This suggests that the promoter context, and perhaps the presence or absence of corepressor complexes associated with promoters in the inactive state (32), may determine which NF-␬B-activated promoters are repressed by fenofibrate. In addition to repressing TNF-␣-mediated activation of the CXCL10 promoter, fenofibrate also repressed activation of the promoter by IFN␥. This repression very likely acts via IFN response factor-containing complexes that bind to the IFN-stimulated response element of the CXCL10 promoter after stimulation with IFN␥ (30). Even if the inhibitory role of PPAR␣ agonists on the secretion of chemokines stimulated by IFN␥ (30) and/or TNF-␣ (7) has been proved, their modulatory role on spontaneously secreted chemokines remains to be investigated. PPAR␥ ligands show an inhibitory activity, too, on CXC chemokines, confirming the results of our previous studies (25, 26). However, the relative potency of PPAR␣ agonists on the inhibition of the secretion of CXC chemokine is higher than that of PPAR␥ ligands. This may suggest that PPAR␣ agonists act, at least in part, through different pathways than PPAR␥ agonists. Regarding the mechanism of these actions, PPAR␥ activators may act differently: first, by decreasing CXCL10 promoter activity and inhibiting protein binding to the two NF-␬B sites (37); and second, thiazolidinedione reduce CXCL10 protein levels in a dose-dependent manner at concentrations (nanomolar) that did not affect mRNA levels or NF-␬B activation (38). It has been shown recently that the thiazolidinedione effect is not only mediated by activating the NF-␬B and Stat1 classic pathways, but also involves a rapid increase in phosphorylation and activation of extracellular signal-regulated kinases (ERK1/2) (39). The different effects of PPAR␣ and PPAR␥ agonists on the secretion of chemokines in thyroid cells may be, at least in part, explained by the different involved pathways. This hypothesis is in agreement with the results of other studies in different cells. Nie et al. (40) reported that the PPAR␥ agonists 15-deoxy-12,14-prostaglandin 2 (15d-PGJ2) and troglitazone, but not PPAR␣ agonist WY-14643, inhibited TNF-induced production of eotaxin and monocyte chemotactic protein-1. The presence of PPAR␣ in the thyroid cells and the immunomodulatory effect of PPAR␣ agonists on the pro-

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duction of Th1 chemokines suggest that PPAR␣ may be involved in the modulation of the immune response both in control and in GD cells, probably by interaction with endogenous ligands. A therapeutic use of PPAR␣ ligands in thyroid autoimmunity remains to be explored, but it is suggested by the fact that their effect on chemokines has been exerted at near-therapeutic doses. If the immunomodulatory role of PPAR␣ ligands will be confirmed in orbital fibroblasts and preadipocytes, a therapeutic use of PPAR␣ ligands in Graves’ orbitopathy might be hypothesized. In conclusion, the present study shows the presence of PPAR␣ in primary normal thyrocytes such as in those from patients with GD. PPAR␣ activators are able to inhibit the secretion of the CXCL9 and CXCL11 chemokines, suggesting that PPAR␣ may be involved in the modulation of the immune response in the thyroid.

Acknowledgments Address all correspondence and requests for reprints to: Alessandro Antonelli, M.D., Department of Internal Medicine, University of Pisa School of Medicine, Via Roma, 67, I-56100, Pisa, Italy. E-mail: [email protected]. Disclosure Summary: The authors have nothing to disclose.

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11.

12.

13.

14.

15.

16.

17.

References 1. Kota BP, Huang TH, Roufogalis BD 2005 An overview on biological mechanisms of PPARs. Pharmacol Res 51:85–94 2. Berger JP, Akiyama TE, Meinke PT 2005 PPARs: therapeutic targets for metabolic disease. Trends Pharmacol Sci 26:244 –251 3. Michalik L, Auwerx J, Berger JP, Chatterjee VK, Glass CK, Gonzalez FJ, Grimaldi PA, Kadowaki T, Lazar MA, O’Rahilly S, Palmer CN, Plutzky J, Reddy JK, Spiegelman BM, Staels B, Wahli W 2006 International Union of Pharmacology. LXI. Peroxisome proliferator-activator receptors. Pharmacol Rev 58:726 –741 4. Kielian T, Drew PD 2003 Effects of peroxisome proliferator-activated receptor-␥ agonists on central nervous system inflammation. J Neurosci Res 71:315–325 5. Lovett-Racke AE, Hussain RZ, Northrop S, Choy J, Rocchini A, Matthes L, Chavis JA, Diab A, Drew PD, Racke MK 2004 Peroxisome proliferator-activated receptor ␣ agonists as therapy for autoimmune disease. J Immunol 172:5790 –5798 6. Cuzzocrea S, Di Paola R, Mazzon E, Genovese T, Muia` C, Centorrino T, Caputi AP 2004 Role of endogenous and exogenous ligands for the peroxisome proliferators activated receptors ␣ (PPAR-␣) in the development of inflammatory bowel disease in mice. Lab Invest 84:1643– 1654 7. Lee JW, Bajwa PJ, Carson MJ, Jeske DR, Cong Y, Elson CO, Lytle C, Straus DS 2007 Fenofibrate represses interleukin-17 and interferon-␥ expression and improves colitis in interleukin-10 deficient mice. Gastroenterology 133:108 –123 8. Delayre-Orthez C, Becker J, Guenon I, Lagente V, Auwerx J, Frossard N, Pons F 2005 PPAR␣ downregulates airway inflammation induced by lipopolysaccharide in the mouse. Respir Res 6:91 9. Cuzzocrea S, Mazzon E, Dugo L, Patel NS, Serraino I, Di Paola R, Genovese T, Britti D, De Maio M, Caputi AP, Thiemermann C 2003

18.

19.

20.

21.

22.

23.

24.

E419

Reduction in the evolution of murine type II collagen-induced arthritis by treatment with rosiglitazone, a ligand of the peroxisome proliferator-activated receptor ␥. Arthritis Rheum 48:3544 –3556 Okamoto H, Iwamoto T, Kotake S, Momohara S, Yamanaka H, Kamatani N 2005 Inhibition of NF-␬B signaling by fenofibrate, a peroxisome proliferator-activated receptor-␣ ligand, presents a therapeutic strategy for rheumatoid arthritis. Clin Exp Rheumatol 23:323–330 Lo Verme J, Fu J, Astarita G, La Rana G, Russo R, Calignano A, Piomelli D 2005 The nuclear receptor peroxisome proliferator-activated receptor-␣ mediates the anti-inflammatory actions of palmitoyl ethanolamide. Mol Pharmacol 67:15–19 Oliveira AC, Bertollo CM, Rocha LT, Nascimento Jr EB, Costa KA, Coelho MM 2007 Antinociceptive and antiedematogenic activities of fenofibrate, an agonist of PPAR␣, and pioglitazone, an agonist of PPAR␥. Eur J Pharmacol 561:194 –201 Ivashchenko CY, Duan SZ, Usher MG, Mortensen RM 2007 PPAR-␥ knockout in pancreatic epithelial cells abolishes the inhibitory effect of rosiglitazone caerulein-induced acute pancreatitis. Am J Physiol Gastrointest Liver Physiol 293:G319 –G326 Gervois P, Kleemann R, Pilon A, Percevault F, Koenig W, Staels B, Kooistra T 2004 Global suppression of IL-6-induced acute phase response gene expression after chronic in vivo treatment with the peroxisome proliferator-activated receptor-␣ activator fenofibrate. J Biol Chem 279:16154 –16160 Cunard R, Ricote M, DiCampli D, Archer DC, Kahn DA, Glass CK, Kelly CJ 2002 Regulation of cytokine expression by ligands of peroxisome proliferator activated receptors. J Immunol 168:2795– 2802 Delerive P, De Bosscher K, Besnard S, Vanden Berghe W, Peters JM, Gonzalez FJ, Fruchart JC, Tedgui A, Haegeman G, Staels B 1999 Peroxisome proliferator-activated receptor ␣ negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-␬B and AP-1. J Biol Chem 274:32048 – 32054 Delerive P, Gervois P, Fruchart JC, Staels B 2000 Induction of I␬B␣ expression as a mechanism contributing to the anti-inflammatory activities of peroxisome proliferator-activated receptor-activators. J Biol Chem 275:36703–36707 Romagnani P, Rotondi M, Lazzeri E, Lasagni L, Francalanci M, Buonamano A, Milani S, Vitti P, Chiovato L, Tonacchera M, Bellastella A, Serio M 2002 Expression of IP-10/CXCL10 and MIG/ CXCL9 in the thyroid and increased levels of IP-10/CXCL10 in the serum of patients with recent-onset Graves’ disease. Am J Pathol 161:195–206 Antonelli A, Rotondi M, Fallahi P, Romagnani P, Ferrari SM, Buonamano A, Ferrannini E, Serio M 2004 High levels of circulating CXCL10 are associated with chronic autoimmune thyroiditis and hypothyroidism. J Clin Endocrinol Metab 89:5496 –5499 Antonelli A, Rotondi M, Fallahi P, Romagnani P, Ferrari SM, Paolicchi A, Ferrannini E, Serio M 2005 Increase of interferon-␥ inducible ␣ chemokine CXCL10 but not ␤ chemokine CCL2 serum levels in chronic autoimmune thyroiditis. Eur J Endocrinol 152: 171–177 García-Lo´pez MA, Sancho D, Sa´nchez-Madrid F, Marazuela M 2001 Thyrocytes from autoimmune thyroid disorders produce the chemokines IP-10 and Mig and attract CXCR3⫹ lymphocytes. J Clin Endocrinol Metab 86:5008 –5016 Kemp EH, Metcalfe RA, Smith KA, Woodroofe MN, Watson PF, Weetman AP 2003 Detection and localization of chemokine gene expression in autoimmune thyroid disease. Clin Endocrinol (Oxf) 59:207–213 Antonelli A, Fallahi P, Rotondi M, Ferrari SM, Serio M, Miccoli P 2006 Serum levels of the interferon-␥-inducible ␣ chemokine CXCL10 in patients with active Graves’ disease, and modulation by methimazole therapy and thyroidectomy. Br J Surg 93:1226 –1231 Antonelli A, Rotondi M, Fallahi P, Grosso M, Boni G, Ferrari SM, Romagnani P, Serio M, Mariani G, Ferrannini E 2007 Iodine-131

CXCL9, CXCL11, PPAR␣ in GD Thyrocytes

J Clin Endocrinol Metab, December 2010, 95(12):E413–E420

given for therapeutic purposes modulates differently interferon-␥inducible ␣-chemokine CXCL10 serum levels in patients with active Graves’ disease or toxic nodular goiter. J Clin Endocrinol Metab 92:1485–1490 Antonelli A, Rotondi M, Ferrari SM, Fallahi P, Romagnani P, Franceschini SS, Serio M, Ferrannini E 2006 Interferon-␥-inducible ␣-chemokine CXCL10 involvement in Graves’ ophthalmopathy: modulation by peroxisome proliferator-activated receptor-␥ agonists. J Clin Endocrinol Metab 91:614 – 620 Antonelli A, Ferrari SM, Fallahi P, Frascerra S, Santini E, Franceschini SS, Ferrannini E 2009 Monokine induced by interferon ␥ (IFN␥) (CXCL9) and IFN␥ inducible T-cell ␣-chemoattractant (CXCL11) involvement in Graves’ disease and ophthalmopathy: modulation by peroxisome proliferator-activated receptor-␥ agonists. J Clin Endocrinol Metab 94:1803–1809 Kasai K, Banba N, Hishinuma A, Matsumura M, Kakishita H, Matsumura M, Motohashi S, Sato N, Hattori Y 2000 15-DeoxyDelta(12,14)-prostaglandin J(2) facilitates thyroglobulin production by cultured human thyrocytes. Am J Physiol Cell Physiol 279: C1859 –C1869 Livak KJ, Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402– 408 Xu J, Chavis JA, Racke MK, Drew PD 2006 Peroxisome proliferatoractivated receptor-␣ and retinoid X receptor agonists inhibit inflammatory responses of astrocytes. J Neuroimmunol 176:95–105 Majumder S, Zhou LZ, Chaturvedi P, Babcock G, Aras S, Ransohoff RM 1998 p48/STAT-1 ␣-containing complexes play a predominant role in induction of IFN-␥-inducible protein, 10kDA (IP-10) by IFN-␥ alone or in synergy with TNF-␣. J Immunol 161:4736 – 4744 Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK 1998 The peroxisome proliferator-activated receptor-␥ is a negative regulator of macrophage activation. Nature 391:79 – 82 Pascual G, Fong AL, Ogawa S, Gamliel A, Li AC, Perissi V, Rose DW, Willson TM, Rosenfeld MG, Glass CK 2005 A SUMOylationdependent pathway mediates transrepression of inflammatory response genes by PPAR-␥. Nature 437:759 –763

33. Leung TH, Hoffmann A, Baltimore D 2004 One nucleotide in a ␬B site can determine cofactor specificity for NF-␬B dimers. Cell 118: 453– 464 34. Kwon JH, Keates S, Simeonidis S, Grall F, Libermann TA, Keates AC 2003 ESE-1, an enterocyte-specific Ets transcription factor, regulates MIP-3␣ gene expression in Caco-2 human colonic epithelial cells. J Biol Chem 278:875– 884 35. Wu GD, Lai EJ, Huang N, Wen X 1997 Oct-1 and CCAAT/enhancer-binding protein (C/EBP) bind to overlapping elements within the interleukin-8 promoter. The role of Oct-1 as a transcriptional repressor. J Biol Chem 272:2396 –2403 36. Wood LD, Richmond A 1995 Constitutive and cytokine-induced expression of the melanoma growth stimulatory activity/GRO ␣ gene requires both NF-␬B and novel constitutive factors. J Biol Chem 270:30619 –30626 37. Marx N, Mach F, Sauty A, Leung JH, Sarafi MN, Ransohoff RM, Libby P, Plutzky J, Luster AD 2000 Peroxisome proliferator-activated receptor-␥ activators inhibit IFN-␥-induced expression of the T cell-active CXC chemokines IP-10, Mig, and I-TAC in human endothelial cells. J Immunol 164:6503– 6508 38. Schaefer KL, Denevich S, Ma C, Cooley SR, Nakajima A, Wada K, Schlezinger J, Sherr D, Saubermann LJ 2005 Intestinal antiinflammatory effects of thiazolidinedione peroxisome proliferator-activated receptor-␥ ligands on T helper type 1 chemokine regulation include nontranscriptional control mechanisms. Inflamm Bowel Dis 11:244 –252 39. Lombardi A, Cantini G, Piscitelli E, Gelmini S, Francalanci M, Mello T, Ceni E, Varano G, Forti G, Rotondi M, Galli A, Serio M, Luconi M 2008 A new mechanism involving ERK contributes to rosiglitazone inhibition of tumor necrosis factor-␣ and interferon-␥ inflammatory effects in human endothelial cells. Arterioscler Thromb Vasc Biol 28:718 –724 40. Nie M, Corbett L, Knox AJ, Pang L 2005 Differential regulation of chemokine expression by peroxisome proliferator-activated receptor ␥ agonists: interactions with glucocorticoids and ␤2-agonists. J Biol Chem 280:2550 –2561

E420

25.

26.

27.

28.

29.

30.

31.

32.

Antonelli et al.