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Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice
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Joo Yeon Jhun a,1 , Seung Hoon Lee a,1 , Jae-Kyeong Byun a , Jeong-Hee Jeong b , Eun-Kyung Kim a , Jennifer Lee a,c , Young-Ok Jung d , Dongyun Shin e , Sung Hwan Park a,c,∗,2 , Mi-La Cho a,c,∗∗,2
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The Rheumatism Research Center, Catholic Research Institute of Medical Science, The Catholic University of Korea, Seoul, South Korea Impact Biotech, Korea, 505 Banpo-Dong, Seocho-Ku, Seoul 137-040, South Korea c Divison of Rheumatology, Department of Internal Medicine, The Catholic University of Korea, Seoul 137-040, South Korea d Kangnam Sacred Heart Hospital and Hallym University, Seoul, South Korea e College of Pharmacy, Gachon University, Incheon, South Korea b
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Article history: Received 10 April 2015 Received in revised form 23 May 2015 Accepted 24 May 2015 Available online xxx
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Keywords: Coenzyme Q10 Rheumatoid arthritis Joint inflammation
Coenzyme Q10 (CoQ10) is a lipid-soluble antioxidant synthesized in human body. This enzyme promotes immune system function and can be used as a dietary supplement. Rheumatoid arthritis (RA) is an autoimmune disease leading to chronic joint inflammation. RA results in severe destruction of cartilage and disability. This study aimed to investigate the effect of CoQ10 on inflammation and Th17 cell proliferation on an experimental rheumatoid arthritis (RA) mice model. CoQ10 or cotton seed oil as control was orally administrated once a day for seven weeks to mice with zymosan-induced arthritis (ZIA). Histological analysis of the joints was conducted using immunohistochemistry. Germinal center (GC) B cells, Th17 cells and Treg cells of the spleen tissue were examined by confocal microscopy staining. mRNA expression was measured by real-time PCR and protein levels were estimated by enzyme-linked immunosorbent assay (ELISA). Flow cytometric analysis (FACS) was used to evaluate Th17 cells and Treg cells. CoQ10 mitigated the severity of ZIA and decreased serum immunoglobulin concentrations. CoQ10 also reduced RANKL-induced osteoclastogenesis, inflammatory mediators and oxidant factors. Th17/Treg axis was reciprocally controlled by CoQ10 treatment. Moreover, CoQ10 treatment on normal mouse and human cells cultured in Th17 conditions decreased the number of Th17 cells and enhanced the number of Treg cells. CoQ10 alleviates arthritis in mice with ZIA declining inflammation, Th17 cells and osteoclast differentiation. These findings suggest that CoQ10 can be a potential therapeutic substance for RA. © 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction
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∗ Corresponding author at: Division of Rheumatology, Department of Internal Medicine, School of Medicine, The Catholic University of Korea, Seoul St. Mary’s Hospital, 505 Banpo-dong, Seocho-gu, Seoul 137-701, South Korea. Tel.: +82 2 2258 6011; fax: +82 2 599 3589. ∗∗ Corresponding author at: Conversant Research Consortium in Immunologic Disease, College of Medicine, The Catholic University of Korea, 505 Banpo-Dong, Seocho-Ku, Seoul 137-040, South Korea. Tel.: +82 2 2258 7467; fax: +82 2 599 4287. E-mail addresses: [email protected] (J.Y. Jhun), [email protected] (S.H. Lee), [email protected] (J.-K. Byun), [email protected] (J.-H. Jeong), [email protected] (E.-K. Kim), [email protected] (J. Lee), [email protected] (Y.-O. Jung), [email protected] (D. Shin), [email protected] (S.H. Park), [email protected] (M.-L. Cho). 1 Joo Yeon Jhun and Seung Hoon Lee contributed equally to this work. 2 Sung Hwan Park and Mi-La Cho contributed equally to this work.
Rheumatoid arthritis (RA) is a systemic autoimmune disease that can cause chronic joint inflammation causing destruction of the adjacent cartilage and disability. Though the exact etiology of the RA pathogenesis is not clear, the up-regulation of inflammatory cytokines such as interleukin (IL)-17 plays a significant role in RA pathogenesis. For instance, the expression of IL-17 increased in patients with RA compared to healthy controls [1]. Th17 cells revealed IL-17 could lead to chronic destructive disor- Q5 der and inflammation in RA patients [2]. There are several reports stating that the IL-17 is therapeutic target in RA and the inhibition of IL-17 reduces Th17 cell activity in RA patients [3,4]. It is also known that the redox imbalance is supposed to play a factor in the pathology of RA. Several investigations have reported Q6 that oxidative stress is involved in the pathogenesis of RA [5,6]. It has been well established that free radicals and reactive oxygen species are involved with the pathogenesis of RA [7,8]. There is also
http://dx.doi.org/10.1016/j.imlet.2015.05.012 0165-2478/© 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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evidence showing increased levels of oxidative stress among RA patients when compared to controls [9] and the balance between reactive oxygen species production and the antioxidant defense system is disproportionate in RA [10]. Antioxidants perform a significant role in anti-inflammatory mechanism. As oxidative stress can lead to inflammatory response [11], antioxidants reduce inflammation preventing the generation of toxic oxidants [12]. It has been suggested that antioxidants are used to suppress inflammation [13,14]. Antioxidants are also related with RA treatment. It has been suggested that antioxidant therapy for the treatment of RA has the possibility of showing a meaningful relief of clinical symptoms [15,16]. Coenzyme Q10 (CoQ10), also known asubidecarenone, is an oil-soluble substance and is present in most eukaryotic cells. This enzyme reduced oxidative stress and partakes in the oxidative phosphorylation pathway. It is well documented that CoQ10 attenuated oxidative stress induced by injury [17,18]. It has also been documented that an important role of CoQ10 is to generate adenosine triphosphate (ATP), which are involved in oxidative phosphorylation pathway within the mitochondrias [19]. Furthermore, CoQ10 has shown to suppress inflammation and arthritis where treatment induced an antiinflammatory response, anti-arthritic and anti-oxidative effect [20,21]. We hypothesized that CoQ10 has an inhibitory effect on the inflammatory response in RA. The aim of the present study was to determine whether CoQ10 induces an anti-inflammatory and anti-arthritic effect and have a therapeutic function in RA mice. Thus, we attempted to illuminate the therapeutic function of CoQ10 in RA underlying its inhibition of IL-17 and Th17 cells mediated inflammation in a mice model of RA.
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2. Materials and methods
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2.1. Animals
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SKG mice with the BALB/c background were kindly obtained from Professor Shimon Sakaguchi (Department of Experimental Immunology, World Premier International Immunology Frontier Research Center, Osaka University). The mice were maintained in a specific pathogen-free environment under climate-controlled conditions with a 12 h-light/dark cycle at the Catholic University of Korea. They were fed standard mouse chow (Ralston Purina, St. Louis, MO, USA) and water ad libitum. All surgeries were performed under isoflurane anesthesia and all efforts were conducted to minimize suffering. All experimental procedures were performed and approved by the Institutional Animal Care and Use Committee (IACUC) at the School of Medicine, Animal Research Ethics Committee of the Catholic University of Korea and were conducted in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals. This animal care and use protocol was reviewed and approved by the Catholic University of Korea.
visually two times per week for the appearance of arthritis in the peripheral joints. 2.3. Measurement of IgG Anti-Total IgG, IgG1, and IgG2a were measured by mouse Total IgG, IgG1, and IgG2a ELISA quantitation kits (Bethyl Laboratories, Montgomery, TX). 2.4. Clinical scoring of arthritis The severity of arthritis was recorded using the mean arthritis index on scale of 0–4, as previously reported [22], as follows: (0), no evidence of erythema and swelling; (1), erythema and mild swelling confined to the mid foot (tarsals) or ankle joint; (2), erythema and mild swelling extending from the ankle to the mid foot; (3), erythema and moderate swelling extending from the ankle to the metatarsal joints; (4), erythema and severe swelling encompassing the ankle, foot, and digits. The severity of arthritis was analyzed by the sum of scores from all legs, assessed by two independent observers with no knowledge of the experimental groups. 2.5. Real-time quantitative polymerase chain reaction (PCR) The mRNA expression levels were estimated using a Light Cycler 2.0 instrument (Roche Diagnostic, Mannheim, Germany) with the version 4.0 software. All reactions were performed with the Light Cycler FastStart DNA Master SYBR Green I (Takara, Shiga, Japan), following the manufacturer’s instructions. The mRNA expression was normalized to that of -actin. The primers sequences are shown in Table 1. Q7 2.6. Osteoclast differentiation assay Osteoclasts cultured in MEM supplemented with 10% fetal bovine serum were stimulated in the presence of macrophage colony-stimulating factor (M-CSF) (10 ng/ml) (R&D Systems) and receptor activator of nuclear factor kappa-B ligand (RANKL) (50 ng/ml) (PeproTech, London, UK) and absence or presence of CoQ10. The medium was changed every two days. Osteoclasts were generated after 8–10 days. 2.7. TRAP staining A commercial TRAP kit (Sigma, St Louis, ML, USA) was used according to the manufacturer’s instructions; however, counterstaining with hematoxylin was omitted. TRAP-positive multinuclear cells (MNCs) containing three or more nuclei were counted as osteoclasts. 2.8. Mixed leukocyte reaction
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2.2. Induction of arthritis and CoQ10 treatment ZIA was induced in SKG mice (n = 20). Zimosan A (Sigma, St Louis, MO, USA) was suspended in phosphate-buffered saline (PBS) and incubated for 10 min in boiling water. Then the zymosan A solution (2 mg/mice) was injected intraperitoneally (I.P.) into 7- or 8-weekold mice. ZIA mice were orally fed once a day for 7 weeks with 20 mg/kg CoQ10 or cotton seed oil as a control beginning on day 7 after first immunization. Arthritis in these mice was examined
Splenocytes were obtained from vehicle and CoQ10 treated mice and culture in RPMI 1640 supplemented with 5% fetal bovine serum. A single suspension was prepared, and 2 × 105 cells/well in 96 well flat bottom plates were cultured in the absence or presence of plate-bound anti-CD3 0.5 g/ml at 37 ◦ C for 72 h, followed by the incorporation of 25 Ci/ml [3 H]-thymidine (GE Healthcare, Piscataway, NJ) for the last 16 h of the indicated total culture interval. Then the radioactivity was measured with a Micro Beta (Pharmacia Biotech, Piscataway, NJ)
Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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Fig. 1. Therapeutic effects of CoQ10 in ZIA mice model. ZIA was induced in SKG mice. CoQ10 (20 mg/kg) or cotton seed oil was orally fed one time in everyday into ZIA induced SKG mice. Mice were sacrificed on day 35 after first immunization. (A) Clinical scores in ZIA induced SKG mice (*P < 0.05, n = 10). (B) The joint tissues from ZIA: CoQ10 treated ZIA mice were stained with H&E (original magnification, 40×, n = 6), safranin O, and toluidine blue (original magnification, 200×, n = 6). (C and D) Immunohistochemical detection of IL-21, -17, IL-1, TNF-␣, VEGF, nitrotyrosine and iNOS was stained in the synovium of ZIA and CoQ10 treated ZIA. All tissues were counterstained with hematoxylin (original magnification, 400×, n = 6). All histological analyses were conducted more than 3 times and representative images are shown. (E) T cell activity of ZIA and CoQ10 ZIA was measured by T cell proliferative respond assay. Data are presented as the mean ± SD of three independent experiments (*P < 0.05).
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2.9. Human CD4+ T cell isolation and differentiation CD4+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) by a CD4+ T cell isolation kit (Miltenyi
Biotec) according to the manufacturer’s protocol. The CD4+ T cells were stimulated with plate-bound anti-CD3, anti-CD28, anti-IFN-␥, anti-IL-4, IL-1 (20 ng/ml) and IL-6 (20 ng/ml) for 3 days to confirm Th17 cell-polarizing conditions. All cytokines were purchased
Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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Q12 Fig. 2. CoQ10 treatment inhibits osteoclastogenesis in ZIA mice. (A) The number of TRAP+ cells in the arthritic joint of ZIA or CoQ10 treated ZIA was counted using light microscopy (n = 6). (B) The BMM cells from the SKG mice were cultured with M-CSF (10 ng/ml) and RANKL (50 ng/ml) with or without various concentrations of CoQ10. Cells were fixed and stained for TRAP (original magnification, 100×). The number of TRAP+ cells was counted using light microscopy. The representative photographs from each group are shown. Data are presented as the mean ± SD of three independent experiments (*P < 0.05, n = 6).
hematoxylin and eosin, Safranin O, and toluidine blue to detect proteoglycans.
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from R&D Systems. Informed consent was obtained from all subjects according to the Declaration of Helsinki. Approval by the ethics committee of Seoul St. Mary’s Hospital (Seoul, Republic of Korea) was obtained from all procedures.
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2.10. Enzyme-linked immunosorbent assay (ELISA)
Spleen tissues of CoQ10 treated mice and vehicle treated mice were obtained on day 35 after first immunization. The tissue was stained using PE-conjugated anti-CD4 and anti-GL-7, FITC-conjugated anti-CD4, anti-GL-7, anti-PD-1 and anti-forkhead box P3 (FOXP3), PE-conjugated anti-B220, anti-CD4 and antiCD25, Cy3-conjugated anti-CD4, anti-pSTAT3 (Y705), anti-pSTAT3 (S727), anti-pSTAT5, and anti-ICOS, B220 (all from eBiosciences, San Diego, CA, USA). Stained sections were analyzed using a Zeiss microscope (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany).
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The supernatant was collected 3 days after CoQ10 treatment. The amount of IL-17, IL-21and TNF-␣ was measured using a sandwich ELISA (R&D Systems). Absorbance at 405 nm was measured using an ELISA microplate reader (Molecular Devices). 2.11. Immunohistochemistry Immunohistochemistry was performed using the Vectastatin ABC kit (Vector Laboratories, Burlingame, CA, USA). Joint tissue of CoQ10 treated mice and vehicle treated mice was first incubated with primary antibodies to IL-21, IL-1, IL-17, TNF␣, VEGF, Nitrotyrosine (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and iNOS (Abcam, Cambridge Science Park, Cambridge, UK) overnight at 4 ◦ C. The sections were counterstained with hematoxylin. Samples were photographed with an Olympus photomicroscope (Tokyo, Japan). Mouse joint tissue was fixed in 4% paraformaldehyde, decalcification EDTA bone decalcifier and embedded in paraffin. The section (7 m) was stained with
2.12. Confocal microscopy
2.13. Flow cytometry To analyze intracellular cytokines, splenocytes cultured in Th17 condition were stained with PerCP-conjugated anti-CD4, APCconjugated anti-CD25, PE-conjugated anti-IL-17A, FITC-conjugated anti-IFN-␥ and FITC-conjugated anti-forkhead box P3 (Foxp3), followed by fixation and permeabilization with a Foxp3 staining buffer kit (BD Bioscience) according to the manufacturer’s instructions. Four hours before the staining, the cells were stimulated with
Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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Q13 Fig. 3. CoQ10 treatment reduces autoantibodies, GC B cells and Tfh cells. (A) ZIA Mice serum was obtained on day 35 after first immunization. The levels of IgG, IgG1, and Q14 IgG2a antibodies were measured from each group. Mean ± SD of three independent experiments (*P < 0.05, n = 10). (B) Spleens of CoQ10 treated ZIA mice or vehicle treated ZIA mice were subjected to confocal staining for CD4+GL-7+CD138+B220+ cells (n = 6). (C) Spleens of CoQ10 treated ZIA mice or vehicle treated ZIA mice were subjected to confocal staining for CD4+GL-7+ICOS+PD-1+ cells (n = 6).
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phorbolmyristate acetate (25 ng/ml) and ionomycin (250 ng/ml) (all from Sigma–Aldrich) and GolgiStop (BD Bioscience). All data were analyzed using Flow Jo software (Tree Star, Ashland, OR, USA).
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All data were expressed as the mean ± standard deviation (SD). Experimental values are presented as mean ± SD of triplicate cultures and representative of experiments performed on three occasions. Statistical significance was determined by Mann–Whitney U test or ANOVA with Bonferroni’s post hoc test using the Graphpad Prism (v.5.01). Values of P < 0.05 were considered statistically significant.
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3. Results
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3.1. CoQ10 attenuates zymosan-induced arthritis
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Mice with ZIA were orally fed with either CoQ10 or cotton seed oil one time a day from day 7 after first immunization to investigate the antiarthritic effect of CoQ10. ZIA mice treated with CoQ10 showed significantly decreased the RA severity (Fig. 1A, left) and
incidence of clinical arthritis (Fig. 1A, right). Histological scores measured based on inflammatory cell infiltration and cartilage damage were markedly lower in CoQ10 treated mice compared to the vehicle (Fig. 1B). Immunohistochemical analysis showed that the CoQ10 treated mice expressed significantly lower levels of proinflammatory cytokines such as IL-21, -1, -17, TNF-␣ and VEGF (Fig. 1C). Also, oxidative stress markers including nitrotyrosine and iNOS expression were significantly reduced in ZIA mice treated with CoQ10 (Fig. 1D). Splenocyte activity was inhibited in CoQ10 treated mice compared to vehicle in normal condition and anti-CD3 condition (Fig. 1E). 3.2. CoQ10 suppresses osteoclastogenesis in ZIA mice To establish CoQ10 effect on osteoclastogenesis, we conducted immunochemical staining for RANK and RANKL in joint tissues of mice. CoQ10 treated ZIA mice revealed decreased bone erosion and cartilage damage in the arthritic joints (Fig. 2A). We performed whether CoQ10 would directly suppress osteoclast formation in vitro. The number of TRAP positive cells was significantly decreased in the joint tissues of CoQ10 treated mice compared with those of the vehicle-treated mice (Fig. 2B).
Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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Fig. 4. CoQ10 treatment decreases Th17 cells. (A) Relative mRNA level of IL-17, CCL20 and ROR␥t was diminished by CoQ10 treatment (*P < 0.05, n = 10). (B) After the isolation of splenocytes from CoQ10 treated ZIA mice or vehicle treated ZIA mice, the populations of IL-17 and IFN-␥ producing CD4+ T cells, and Foxp3 producing C25+ T cells were analyzed by using antibodies specific for CD4, CD25, IFN-␥ and IL-17 by intracellular flow cytometric analysis. Data are presented as the mean ± SD of three independent experiments (*P < 0.05, n = 10).
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3.3. CoQ10 reduces autoantibody, GC B cells and Tfh cells in ZIA mice Total IgG, IgG1, and IgG2a antibody was significantly reduced in ZIA mice treated with CoQ10 when compared to controls (Fig. 3A). Germinal centers (GC) B cells markers including GL-7, CD138 and B220 were decreased in CoQ10 treated mice (Fig. 3B). T follicular helper (Tfh) cells marker such as GL-7, ICOS and PD-1 were also diminished reduced in ZIA mice treated with CoQ10 (Fig. 3C). The number of GC cells and Tfh cells was significantly reduced in CoQ10 treated mice compared to the vehicle-treated mice (Fig. 3B and C).
3.4. CoQ10 decreases IL-17 expression and induces Foxp3 expression Total RNA was isolated from splenocytes of either CoQ10 treated ZIA mice or vehicle treated ZIA mice and the mRNA expression of
Th17 cell and Treg cell related markers by real-time PCR. The results showed that the mRNA levels of IL-17, CCL20 and ROR␥t, Th17 cell-related molecules, were decreased significantly in splenocyte of CoQ10 treated mice (Fig. 4A). In flow cytometry analysis, IFN-␥ and IL-17 expressions were down-regulated in the splenocytes of CoQ10 treated mice. On the other hand, the expression of Treg cell-related molecules such as Foxp3 was up-regulated in CoQ10 treated ZIA mice (Fig. 4B). Furthermore, CoQ10 treatment decreased the number of IL-17 producing CD4+CD25+ T cells in the spleen tissues of ZIA mice as analyzed by immunofluorescence confocal microscopy. CoQ10 treatment induced the numbers of CD4+CD25+Foxp3+ regulatory T cells in tissues of spleens compared with those of the vehicle treatment (Fig. 5A). The spleen tissues from mice treated with CoQ10 revealed a down-regulated number of phosphorylated STAT3 727 and 705+ regulatory T cells and an up-regulated number of phosphorylated STAT5+ cells compared with those of the vehicle-treated mice measured by immunofluorescence confocal microscopy (Fig. 5B).
Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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Fig. 5. CoQ10 treatment enhances Treg cells and down-regulates STAT3 phosphorylation in the CD4+ T cells in mice. (A) Spleens of CoQ10 treated ZIA mice or vehicle-treated ZIA mice were subjected to immunostaining for CD4+IL-17 or CD4+CD25+Foxp3+ cells (*P < 0.05, **P < 0.03, n = 6). (B) Spleens of CoQ10 treated ZIA mice or vehicle-treated ZIA mice were subjected to confocal staining for CD4+pSTAT3y705+, CD4+pSTAT3s727+ or CD4+pSTAT5 cells. The number of cells was counted in four independent quadrants. The expression of various signaling molecules was determined by western blotting. Data are presented as the mean ± SD of three independent experiments (*P < 0.05, **P < 0.03, n = 6).
Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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3.5. CoQ10 increased Foxp3-expressing Treg cells and suppressed IL-17-expressing Th17 cells in mouse spleen and human PBMCs
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Treatment of CoQ10 used in these in vitro experiments with mouse splenocytes cultured in Th17 condition significantly reduced mRNA expression of Th17 cell related factors such as IL-17, CCL20, ROR␥t, BATF and RUNX1 (Fig. 6A). CoQ10 treatment significantly suppressed protein levels of TNF-␣ and -21 in the Th17 conditioned media cultured with mouse splenocytes (Fig. 6B). The expression of IL-17 was decreased in flow cytometry (Fig. 6C). In human PBMCs, mRNA levels of Th17 cell related factors such as IL-17, CCL20, ROR␥t were significantly decreased whereas mRNA level of Treg related molecules such as Foxp3 was markedly increased by CoQ10 treatment (Fig. 6D). Additionally, the expression of proinflammatory cytokines including IL-17, -21 and TNF-␣ were significantly reduced by CoQ10 treatment (Fig. 6E). Subsequent flow cytometry analysis verified that the populations of Th17 and Treg cells were indeed reciprocally regulated where Th17 cells were suppressed and Treg cells were enhanced by treatment with CoQ10 (Fig. 6F).
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4. Discussion
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CoQ10 as an antioxidant has shown to have an antiinflammatory function. However, there is little mechanistic evidence of the effect of CoQ10 on the immune-inflammatory response in RA. Here, we investigated the anti-inflammatory function of CoQ10 on a RA mice model. The most meaningful observation of this study is that CoQ10 down-regulated IL-17 expression and Th17 cells population induced by inflammatory response. A significant suppression in
proinflammatory cytokines including IL-17, -21, TNF-␣, and IL1 was detected in the joints of CoQ10 treated ZIA mice. Several studies have demonstrated that IL-17 is a powerful inducer of IL-21, TNF-␣ and IL-1 [23,24]. Th17 cells also aggravate several autoimmune diseases including RA [25]. It is well documented that Th17 activity and expression of IL-17 exacerbate RA progression [26]. On the other hand, Treg cells expressing Foxp3 are involved in anti-inflammatory response [27,28]. As the inhibition of IL-17 expression and Th17 cells and up-regulation of Treg cells are significant property to suppress inflammation, our results have shown that CoQ10 can lead to function of anti-inflammatory response in autoimmune arthritis. Recently, certain researches have reported that the regulation Q8 of Th17 and Treg cells plays a significant role in RA [29,30]. The activation of STAT5 sustains FOXP3 expression in Treg cells, but the activation of STAT3 induces differentiation of Th17 cells [31]. In this study, the number of Treg cells increased with CoQ10 treatment while Th17 differentiation was inhibited. There has been little information about the control of Th17 and Treg cells by CoQ10. It has been demonstrated that transcription of IL-17 is controlled by competitive binding of pSTAT3 and pSTAT5 [32]. Thus, pSTAT5 is a critical transcriptional factor for Foxp3 regulating differentiation of Treg cells. Our results revealed that while CoQ10 significantly induced pSTAT5+ T cells, pSTAT3+ T cells were reduced by CoQ10 treatment. It seems to be caused by the down-regulation of T cell transcriptional regulators such as ROR␥t, BATF and RUNX1 and the Q9 up-regulation of Foxp3. Hence, CoQ10 function to inhibit pSTAT3 and enhance pSTAT5 may be another efficient mechanism by which CoQ10 controls Th17/Treg balance. Oxidative stress is known to play a key role in RA pathogenesis. It has been reported that CoQ10 decreased iNOS and protected
Fig. 6. CoQ10 treatment reduces Th17 cell differentiation in CD4+ T cells isolated from SKG mice and CD4+ T cells isolated from human peripheral blood mononuclear cells. (A) Real-time reverse transcriptase-polymerase chain reaction of IL-17, CCL20, ROR␥t, BATF and RUNX1 mRNA in CD4+ T cells. (B) The production of TNF- and IL-21 induced by Th17 condition in the presence or absence of CoQ10. (C) After CoQ10 treatment, the populations of IL-17 and Foxp3 producing CD4+ T cells were analyzed by using antibodies specific for CD4, Foxp3 and IL-17 by intracellular flow cytometric analysis. (D) Real-time reverse transcriptase-polymerase chain reaction of IL-17, CCL20, RORc, Q15 and FoxP3 mRNA in CD4+ T cells. (E) The production of IL-17, -21 and TNF-␣ induced by Th17 condition in the presence or absence of CoQ10. (F) After several concentration of CoQ10 treatment, the populations of IL-17 and Foxp3 producing CD4+ T cells were analyzed by using antibodies specific for CD4, Foxp3 and IL-17 by intracellular flow cytometric analysis. Data are presented as the mean ± SD of three independent experiments (*P < 0.05, **P < 0.03, ***P < 0.001).
Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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endothelial cells from oxidative stress-induced injury [33]. More recently, CoQ10 acted as a suppressor of iNOS in the joints of osteoarthritis mice [34]. Our data show that the expression of iNOS was reduced after CoQ10 treatment in RA mice joints. As the expression of iNOS and nitrotyrosine indicates oxidative stress, down-regulating the expression of iNOS and nitrotyrosine in the joints of CoQ10 treated mice demonstrated a decrease in the oxidative stress compared to the vehicle treated mice. The inhibitor of Breg cells proliferation was reported as a suppressive factor of arthritis severity in RA murine model [35]. In this study, GCB cells expressing GL-7 are involved with the B
cell immune response and Tfh cells revealing ICOS and PD-1 are involved in the germinal center responses. GC B cells and Tfh cells produce memory B cells and long-lived plasma cells [36]. Our data showed that total IgG, IgG1 and IgG2a level reduced by CoQ10 may cause to inhibit differentiation of GCB cells and Tfh cells. These results suggest that the function of CoQ10 is involved in declining the B cell immune response. The reduction of osteoclastogenesis by CoQ10 treatment was revealed by both in vivo and in vitro. The number of TRAP+ multinucleated cells decreased in the joints of CoQ10 treated mice seems to have resulted in decreased expression of inflammatory cytokines
Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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that enhance osteoclastogenesis through the up-regulation of RANKL in the arthritic joint. It is well reported that the inhibition of osteoclastogenesis suppresses joint inflammation and severity of autoimmune arthritis [37]. Thus, CoQ10 can reveal therapeutic effect in autoimmune arthritis through inhibition of osteoclastogenesis. To our knowledge, the present study provides the first evidence that CoQ10 can regulate Th17 differentiation. By inhibiting STAT3 phosphorylation, CoQ10 concurrently suppresses Th17 and enhances Treg. Furthermore, it can be expected that CoQ10 may have therapeutic potential in other diseases where Th17 plays a major role in the pathogenesis of such as inflammatory bowel disease or graft-versus-host disease. Future research on this topic will be promising. There is little information in the investigation regarding control of Th17 cells by CoQ10. The present study suggests that CoQ10 can regulate Th17 differentiation by inhibiting pSTAT3 while enhancing differentiation of Treg cells via pSTAT5. The CoQ10 functions newly identified in this investigation indicate that it likely performs an important role in attenuating inflammation and may shed light on the pathogenesis of immune-mediated inflammatory diseases including RA. Moreover, CoQ10 can be expected to conduct a significant role in the selection of immune-mediated inflammatory therapeutic targets. This novel evidence suggests that CoQ10 might be a strong candidate for the treatment of RA.
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Competing interests
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The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgements
This study was supported by Research Fund of Seoul St. Mary’s Hospital, The Catholic University of Korea. This research was sup380 Q11 ported by the Bio & Medical Technology Development Program 381 of the National Research Foundation (NRF) funded by the Korean 382 government (MEST) (No. 2012M3A9C6049783). 383 Q10 379
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Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice
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Joo Yeon Jhun a,1 , Seung Hoon Lee a,1 , Jae-Kyeong Byun a , Jeong-Hee Jeong b , Eun-Kyung Kim a , Jennifer Lee a,c , Young-Ok Jung d , Dongyun Shin e , Sung Hwan Park a,c,∗,2 , Mi-La Cho a,c,∗∗,2
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The Rheumatism Research Center, Catholic Research Institute of Medical Science, The Catholic University of Korea, Seoul, South Korea Impact Biotech, Korea, 505 Banpo-Dong, Seocho-Ku, Seoul 137-040, South Korea c Divison of Rheumatology, Department of Internal Medicine, The Catholic University of Korea, Seoul 137-040, South Korea d Kangnam Sacred Heart Hospital and Hallym University, Seoul, South Korea e College of Pharmacy, Gachon University, Incheon, South Korea b
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Article history: Received 10 April 2015 Received in revised form 23 May 2015 Accepted 24 May 2015 Available online xxx
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Keywords: Coenzyme Q10 Rheumatoid arthritis Joint inflammation
Coenzyme Q10 (CoQ10) is a lipid-soluble antioxidant synthesized in human body. This enzyme promotes immune system function and can be used as a dietary supplement. Rheumatoid arthritis (RA) is an autoimmune disease leading to chronic joint inflammation. RA results in severe destruction of cartilage and disability. This study aimed to investigate the effect of CoQ10 on inflammation and Th17 cell proliferation on an experimental rheumatoid arthritis (RA) mice model. CoQ10 or cotton seed oil as control was orally administrated once a day for seven weeks to mice with zymosan-induced arthritis (ZIA). Histological analysis of the joints was conducted using immunohistochemistry. Germinal center (GC) B cells, Th17 cells and Treg cells of the spleen tissue were examined by confocal microscopy staining. mRNA expression was measured by real-time PCR and protein levels were estimated by enzyme-linked immunosorbent assay (ELISA). Flow cytometric analysis (FACS) was used to evaluate Th17 cells and Treg cells. CoQ10 mitigated the severity of ZIA and decreased serum immunoglobulin concentrations. CoQ10 also reduced RANKL-induced osteoclastogenesis, inflammatory mediators and oxidant factors. Th17/Treg axis was reciprocally controlled by CoQ10 treatment. Moreover, CoQ10 treatment on normal mouse and human cells cultured in Th17 conditions decreased the number of Th17 cells and enhanced the number of Treg cells. CoQ10 alleviates arthritis in mice with ZIA declining inflammation, Th17 cells and osteoclast differentiation. These findings suggest that CoQ10 can be a potential therapeutic substance for RA. © 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction
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∗ Corresponding author at: Division of Rheumatology, Department of Internal Medicine, School of Medicine, The Catholic University of Korea, Seoul St. Mary’s Hospital, 505 Banpo-dong, Seocho-gu, Seoul 137-701, South Korea. Tel.: +82 2 2258 6011; fax: +82 2 599 3589. ∗∗ Corresponding author at: Conversant Research Consortium in Immunologic Disease, College of Medicine, The Catholic University of Korea, 505 Banpo-Dong, Seocho-Ku, Seoul 137-040, South Korea. Tel.: +82 2 2258 7467; fax: +82 2 599 4287. E-mail addresses: [email protected] (J.Y. Jhun), [email protected] (S.H. Lee), [email protected] (J.-K. Byun), [email protected] (J.-H. Jeong), [email protected] (E.-K. Kim), [email protected] (J. Lee), [email protected] (Y.-O. Jung), [email protected] (D. Shin), [email protected] (S.H. Park), [email protected] (M.-L. Cho). 1 Joo Yeon Jhun and Seung Hoon Lee contributed equally to this work. 2 Sung Hwan Park and Mi-La Cho contributed equally to this work.
Rheumatoid arthritis (RA) is a systemic autoimmune disease that can cause chronic joint inflammation causing destruction of the adjacent cartilage and disability. Though the exact etiology of the RA pathogenesis is not clear, the up-regulation of inflammatory cytokines such as interleukin (IL)-17 plays a significant role in RA pathogenesis. For instance, the expression of IL-17 increased in patients with RA compared to healthy controls [1]. Th17 cells revealed IL-17 could lead to chronic destructive disor- Q5 der and inflammation in RA patients [2]. There are several reports stating that the IL-17 is therapeutic target in RA and the inhibition of IL-17 reduces Th17 cell activity in RA patients [3,4]. It is also known that the redox imbalance is supposed to play a factor in the pathology of RA. Several investigations have reported Q6 that oxidative stress is involved in the pathogenesis of RA [5,6]. It has been well established that free radicals and reactive oxygen species are involved with the pathogenesis of RA [7,8]. There is also
http://dx.doi.org/10.1016/j.imlet.2015.05.012 0165-2478/© 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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evidence showing increased levels of oxidative stress among RA patients when compared to controls [9] and the balance between reactive oxygen species production and the antioxidant defense system is disproportionate in RA [10]. Antioxidants perform a significant role in anti-inflammatory mechanism. As oxidative stress can lead to inflammatory response [11], antioxidants reduce inflammation preventing the generation of toxic oxidants [12]. It has been suggested that antioxidants are used to suppress inflammation [13,14]. Antioxidants are also related with RA treatment. It has been suggested that antioxidant therapy for the treatment of RA has the possibility of showing a meaningful relief of clinical symptoms [15,16]. Coenzyme Q10 (CoQ10), also known asubidecarenone, is an oil-soluble substance and is present in most eukaryotic cells. This enzyme reduced oxidative stress and partakes in the oxidative phosphorylation pathway. It is well documented that CoQ10 attenuated oxidative stress induced by injury [17,18]. It has also been documented that an important role of CoQ10 is to generate adenosine triphosphate (ATP), which are involved in oxidative phosphorylation pathway within the mitochondrias [19]. Furthermore, CoQ10 has shown to suppress inflammation and arthritis where treatment induced an antiinflammatory response, anti-arthritic and anti-oxidative effect [20,21]. We hypothesized that CoQ10 has an inhibitory effect on the inflammatory response in RA. The aim of the present study was to determine whether CoQ10 induces an anti-inflammatory and anti-arthritic effect and have a therapeutic function in RA mice. Thus, we attempted to illuminate the therapeutic function of CoQ10 in RA underlying its inhibition of IL-17 and Th17 cells mediated inflammation in a mice model of RA.
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SKG mice with the BALB/c background were kindly obtained from Professor Shimon Sakaguchi (Department of Experimental Immunology, World Premier International Immunology Frontier Research Center, Osaka University). The mice were maintained in a specific pathogen-free environment under climate-controlled conditions with a 12 h-light/dark cycle at the Catholic University of Korea. They were fed standard mouse chow (Ralston Purina, St. Louis, MO, USA) and water ad libitum. All surgeries were performed under isoflurane anesthesia and all efforts were conducted to minimize suffering. All experimental procedures were performed and approved by the Institutional Animal Care and Use Committee (IACUC) at the School of Medicine, Animal Research Ethics Committee of the Catholic University of Korea and were conducted in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals. This animal care and use protocol was reviewed and approved by the Catholic University of Korea.
visually two times per week for the appearance of arthritis in the peripheral joints. 2.3. Measurement of IgG Anti-Total IgG, IgG1, and IgG2a were measured by mouse Total IgG, IgG1, and IgG2a ELISA quantitation kits (Bethyl Laboratories, Montgomery, TX). 2.4. Clinical scoring of arthritis The severity of arthritis was recorded using the mean arthritis index on scale of 0–4, as previously reported [22], as follows: (0), no evidence of erythema and swelling; (1), erythema and mild swelling confined to the mid foot (tarsals) or ankle joint; (2), erythema and mild swelling extending from the ankle to the mid foot; (3), erythema and moderate swelling extending from the ankle to the metatarsal joints; (4), erythema and severe swelling encompassing the ankle, foot, and digits. The severity of arthritis was analyzed by the sum of scores from all legs, assessed by two independent observers with no knowledge of the experimental groups. 2.5. Real-time quantitative polymerase chain reaction (PCR) The mRNA expression levels were estimated using a Light Cycler 2.0 instrument (Roche Diagnostic, Mannheim, Germany) with the version 4.0 software. All reactions were performed with the Light Cycler FastStart DNA Master SYBR Green I (Takara, Shiga, Japan), following the manufacturer’s instructions. The mRNA expression was normalized to that of -actin. The primers sequences are shown in Table 1. Q7 2.6. Osteoclast differentiation assay Osteoclasts cultured in MEM supplemented with 10% fetal bovine serum were stimulated in the presence of macrophage colony-stimulating factor (M-CSF) (10 ng/ml) (R&D Systems) and receptor activator of nuclear factor kappa-B ligand (RANKL) (50 ng/ml) (PeproTech, London, UK) and absence or presence of CoQ10. The medium was changed every two days. Osteoclasts were generated after 8–10 days. 2.7. TRAP staining A commercial TRAP kit (Sigma, St Louis, ML, USA) was used according to the manufacturer’s instructions; however, counterstaining with hematoxylin was omitted. TRAP-positive multinuclear cells (MNCs) containing three or more nuclei were counted as osteoclasts. 2.8. Mixed leukocyte reaction
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2.2. Induction of arthritis and CoQ10 treatment ZIA was induced in SKG mice (n = 20). Zimosan A (Sigma, St Louis, MO, USA) was suspended in phosphate-buffered saline (PBS) and incubated for 10 min in boiling water. Then the zymosan A solution (2 mg/mice) was injected intraperitoneally (I.P.) into 7- or 8-weekold mice. ZIA mice were orally fed once a day for 7 weeks with 20 mg/kg CoQ10 or cotton seed oil as a control beginning on day 7 after first immunization. Arthritis in these mice was examined
Splenocytes were obtained from vehicle and CoQ10 treated mice and culture in RPMI 1640 supplemented with 5% fetal bovine serum. A single suspension was prepared, and 2 × 105 cells/well in 96 well flat bottom plates were cultured in the absence or presence of plate-bound anti-CD3 0.5 g/ml at 37 ◦ C for 72 h, followed by the incorporation of 25 Ci/ml [3 H]-thymidine (GE Healthcare, Piscataway, NJ) for the last 16 h of the indicated total culture interval. Then the radioactivity was measured with a Micro Beta (Pharmacia Biotech, Piscataway, NJ)
Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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Fig. 1. Therapeutic effects of CoQ10 in ZIA mice model. ZIA was induced in SKG mice. CoQ10 (20 mg/kg) or cotton seed oil was orally fed one time in everyday into ZIA induced SKG mice. Mice were sacrificed on day 35 after first immunization. (A) Clinical scores in ZIA induced SKG mice (*P < 0.05, n = 10). (B) The joint tissues from ZIA: CoQ10 treated ZIA mice were stained with H&E (original magnification, 40×, n = 6), safranin O, and toluidine blue (original magnification, 200×, n = 6). (C and D) Immunohistochemical detection of IL-21, -17, IL-1, TNF-␣, VEGF, nitrotyrosine and iNOS was stained in the synovium of ZIA and CoQ10 treated ZIA. All tissues were counterstained with hematoxylin (original magnification, 400×, n = 6). All histological analyses were conducted more than 3 times and representative images are shown. (E) T cell activity of ZIA and CoQ10 ZIA was measured by T cell proliferative respond assay. Data are presented as the mean ± SD of three independent experiments (*P < 0.05).
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2.9. Human CD4+ T cell isolation and differentiation CD4+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) by a CD4+ T cell isolation kit (Miltenyi
Biotec) according to the manufacturer’s protocol. The CD4+ T cells were stimulated with plate-bound anti-CD3, anti-CD28, anti-IFN-␥, anti-IL-4, IL-1 (20 ng/ml) and IL-6 (20 ng/ml) for 3 days to confirm Th17 cell-polarizing conditions. All cytokines were purchased
Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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Q12 Fig. 2. CoQ10 treatment inhibits osteoclastogenesis in ZIA mice. (A) The number of TRAP+ cells in the arthritic joint of ZIA or CoQ10 treated ZIA was counted using light microscopy (n = 6). (B) The BMM cells from the SKG mice were cultured with M-CSF (10 ng/ml) and RANKL (50 ng/ml) with or without various concentrations of CoQ10. Cells were fixed and stained for TRAP (original magnification, 100×). The number of TRAP+ cells was counted using light microscopy. The representative photographs from each group are shown. Data are presented as the mean ± SD of three independent experiments (*P < 0.05, n = 6).
hematoxylin and eosin, Safranin O, and toluidine blue to detect proteoglycans.
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from R&D Systems. Informed consent was obtained from all subjects according to the Declaration of Helsinki. Approval by the ethics committee of Seoul St. Mary’s Hospital (Seoul, Republic of Korea) was obtained from all procedures.
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2.10. Enzyme-linked immunosorbent assay (ELISA)
Spleen tissues of CoQ10 treated mice and vehicle treated mice were obtained on day 35 after first immunization. The tissue was stained using PE-conjugated anti-CD4 and anti-GL-7, FITC-conjugated anti-CD4, anti-GL-7, anti-PD-1 and anti-forkhead box P3 (FOXP3), PE-conjugated anti-B220, anti-CD4 and antiCD25, Cy3-conjugated anti-CD4, anti-pSTAT3 (Y705), anti-pSTAT3 (S727), anti-pSTAT5, and anti-ICOS, B220 (all from eBiosciences, San Diego, CA, USA). Stained sections were analyzed using a Zeiss microscope (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany).
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The supernatant was collected 3 days after CoQ10 treatment. The amount of IL-17, IL-21and TNF-␣ was measured using a sandwich ELISA (R&D Systems). Absorbance at 405 nm was measured using an ELISA microplate reader (Molecular Devices). 2.11. Immunohistochemistry Immunohistochemistry was performed using the Vectastatin ABC kit (Vector Laboratories, Burlingame, CA, USA). Joint tissue of CoQ10 treated mice and vehicle treated mice was first incubated with primary antibodies to IL-21, IL-1, IL-17, TNF␣, VEGF, Nitrotyrosine (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and iNOS (Abcam, Cambridge Science Park, Cambridge, UK) overnight at 4 ◦ C. The sections were counterstained with hematoxylin. Samples were photographed with an Olympus photomicroscope (Tokyo, Japan). Mouse joint tissue was fixed in 4% paraformaldehyde, decalcification EDTA bone decalcifier and embedded in paraffin. The section (7 m) was stained with
2.12. Confocal microscopy
2.13. Flow cytometry To analyze intracellular cytokines, splenocytes cultured in Th17 condition were stained with PerCP-conjugated anti-CD4, APCconjugated anti-CD25, PE-conjugated anti-IL-17A, FITC-conjugated anti-IFN-␥ and FITC-conjugated anti-forkhead box P3 (Foxp3), followed by fixation and permeabilization with a Foxp3 staining buffer kit (BD Bioscience) according to the manufacturer’s instructions. Four hours before the staining, the cells were stimulated with
Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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Q13 Fig. 3. CoQ10 treatment reduces autoantibodies, GC B cells and Tfh cells. (A) ZIA Mice serum was obtained on day 35 after first immunization. The levels of IgG, IgG1, and Q14 IgG2a antibodies were measured from each group. Mean ± SD of three independent experiments (*P < 0.05, n = 10). (B) Spleens of CoQ10 treated ZIA mice or vehicle treated ZIA mice were subjected to confocal staining for CD4+GL-7+CD138+B220+ cells (n = 6). (C) Spleens of CoQ10 treated ZIA mice or vehicle treated ZIA mice were subjected to confocal staining for CD4+GL-7+ICOS+PD-1+ cells (n = 6).
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phorbolmyristate acetate (25 ng/ml) and ionomycin (250 ng/ml) (all from Sigma–Aldrich) and GolgiStop (BD Bioscience). All data were analyzed using Flow Jo software (Tree Star, Ashland, OR, USA).
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All data were expressed as the mean ± standard deviation (SD). Experimental values are presented as mean ± SD of triplicate cultures and representative of experiments performed on three occasions. Statistical significance was determined by Mann–Whitney U test or ANOVA with Bonferroni’s post hoc test using the Graphpad Prism (v.5.01). Values of P < 0.05 were considered statistically significant.
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3.1. CoQ10 attenuates zymosan-induced arthritis
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Mice with ZIA were orally fed with either CoQ10 or cotton seed oil one time a day from day 7 after first immunization to investigate the antiarthritic effect of CoQ10. ZIA mice treated with CoQ10 showed significantly decreased the RA severity (Fig. 1A, left) and
incidence of clinical arthritis (Fig. 1A, right). Histological scores measured based on inflammatory cell infiltration and cartilage damage were markedly lower in CoQ10 treated mice compared to the vehicle (Fig. 1B). Immunohistochemical analysis showed that the CoQ10 treated mice expressed significantly lower levels of proinflammatory cytokines such as IL-21, -1, -17, TNF-␣ and VEGF (Fig. 1C). Also, oxidative stress markers including nitrotyrosine and iNOS expression were significantly reduced in ZIA mice treated with CoQ10 (Fig. 1D). Splenocyte activity was inhibited in CoQ10 treated mice compared to vehicle in normal condition and anti-CD3 condition (Fig. 1E). 3.2. CoQ10 suppresses osteoclastogenesis in ZIA mice To establish CoQ10 effect on osteoclastogenesis, we conducted immunochemical staining for RANK and RANKL in joint tissues of mice. CoQ10 treated ZIA mice revealed decreased bone erosion and cartilage damage in the arthritic joints (Fig. 2A). We performed whether CoQ10 would directly suppress osteoclast formation in vitro. The number of TRAP positive cells was significantly decreased in the joint tissues of CoQ10 treated mice compared with those of the vehicle-treated mice (Fig. 2B).
Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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Fig. 4. CoQ10 treatment decreases Th17 cells. (A) Relative mRNA level of IL-17, CCL20 and ROR␥t was diminished by CoQ10 treatment (*P < 0.05, n = 10). (B) After the isolation of splenocytes from CoQ10 treated ZIA mice or vehicle treated ZIA mice, the populations of IL-17 and IFN-␥ producing CD4+ T cells, and Foxp3 producing C25+ T cells were analyzed by using antibodies specific for CD4, CD25, IFN-␥ and IL-17 by intracellular flow cytometric analysis. Data are presented as the mean ± SD of three independent experiments (*P < 0.05, n = 10).
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3.3. CoQ10 reduces autoantibody, GC B cells and Tfh cells in ZIA mice Total IgG, IgG1, and IgG2a antibody was significantly reduced in ZIA mice treated with CoQ10 when compared to controls (Fig. 3A). Germinal centers (GC) B cells markers including GL-7, CD138 and B220 were decreased in CoQ10 treated mice (Fig. 3B). T follicular helper (Tfh) cells marker such as GL-7, ICOS and PD-1 were also diminished reduced in ZIA mice treated with CoQ10 (Fig. 3C). The number of GC cells and Tfh cells was significantly reduced in CoQ10 treated mice compared to the vehicle-treated mice (Fig. 3B and C).
3.4. CoQ10 decreases IL-17 expression and induces Foxp3 expression Total RNA was isolated from splenocytes of either CoQ10 treated ZIA mice or vehicle treated ZIA mice and the mRNA expression of
Th17 cell and Treg cell related markers by real-time PCR. The results showed that the mRNA levels of IL-17, CCL20 and ROR␥t, Th17 cell-related molecules, were decreased significantly in splenocyte of CoQ10 treated mice (Fig. 4A). In flow cytometry analysis, IFN-␥ and IL-17 expressions were down-regulated in the splenocytes of CoQ10 treated mice. On the other hand, the expression of Treg cell-related molecules such as Foxp3 was up-regulated in CoQ10 treated ZIA mice (Fig. 4B). Furthermore, CoQ10 treatment decreased the number of IL-17 producing CD4+CD25+ T cells in the spleen tissues of ZIA mice as analyzed by immunofluorescence confocal microscopy. CoQ10 treatment induced the numbers of CD4+CD25+Foxp3+ regulatory T cells in tissues of spleens compared with those of the vehicle treatment (Fig. 5A). The spleen tissues from mice treated with CoQ10 revealed a down-regulated number of phosphorylated STAT3 727 and 705+ regulatory T cells and an up-regulated number of phosphorylated STAT5+ cells compared with those of the vehicle-treated mice measured by immunofluorescence confocal microscopy (Fig. 5B).
Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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Fig. 5. CoQ10 treatment enhances Treg cells and down-regulates STAT3 phosphorylation in the CD4+ T cells in mice. (A) Spleens of CoQ10 treated ZIA mice or vehicle-treated ZIA mice were subjected to immunostaining for CD4+IL-17 or CD4+CD25+Foxp3+ cells (*P < 0.05, **P < 0.03, n = 6). (B) Spleens of CoQ10 treated ZIA mice or vehicle-treated ZIA mice were subjected to confocal staining for CD4+pSTAT3y705+, CD4+pSTAT3s727+ or CD4+pSTAT5 cells. The number of cells was counted in four independent quadrants. The expression of various signaling molecules was determined by western blotting. Data are presented as the mean ± SD of three independent experiments (*P < 0.05, **P < 0.03, n = 6).
Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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3.5. CoQ10 increased Foxp3-expressing Treg cells and suppressed IL-17-expressing Th17 cells in mouse spleen and human PBMCs
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Treatment of CoQ10 used in these in vitro experiments with mouse splenocytes cultured in Th17 condition significantly reduced mRNA expression of Th17 cell related factors such as IL-17, CCL20, ROR␥t, BATF and RUNX1 (Fig. 6A). CoQ10 treatment significantly suppressed protein levels of TNF-␣ and -21 in the Th17 conditioned media cultured with mouse splenocytes (Fig. 6B). The expression of IL-17 was decreased in flow cytometry (Fig. 6C). In human PBMCs, mRNA levels of Th17 cell related factors such as IL-17, CCL20, ROR␥t were significantly decreased whereas mRNA level of Treg related molecules such as Foxp3 was markedly increased by CoQ10 treatment (Fig. 6D). Additionally, the expression of proinflammatory cytokines including IL-17, -21 and TNF-␣ were significantly reduced by CoQ10 treatment (Fig. 6E). Subsequent flow cytometry analysis verified that the populations of Th17 and Treg cells were indeed reciprocally regulated where Th17 cells were suppressed and Treg cells were enhanced by treatment with CoQ10 (Fig. 6F).
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4. Discussion
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CoQ10 as an antioxidant has shown to have an antiinflammatory function. However, there is little mechanistic evidence of the effect of CoQ10 on the immune-inflammatory response in RA. Here, we investigated the anti-inflammatory function of CoQ10 on a RA mice model. The most meaningful observation of this study is that CoQ10 down-regulated IL-17 expression and Th17 cells population induced by inflammatory response. A significant suppression in
proinflammatory cytokines including IL-17, -21, TNF-␣, and IL1 was detected in the joints of CoQ10 treated ZIA mice. Several studies have demonstrated that IL-17 is a powerful inducer of IL-21, TNF-␣ and IL-1 [23,24]. Th17 cells also aggravate several autoimmune diseases including RA [25]. It is well documented that Th17 activity and expression of IL-17 exacerbate RA progression [26]. On the other hand, Treg cells expressing Foxp3 are involved in anti-inflammatory response [27,28]. As the inhibition of IL-17 expression and Th17 cells and up-regulation of Treg cells are significant property to suppress inflammation, our results have shown that CoQ10 can lead to function of anti-inflammatory response in autoimmune arthritis. Recently, certain researches have reported that the regulation Q8 of Th17 and Treg cells plays a significant role in RA [29,30]. The activation of STAT5 sustains FOXP3 expression in Treg cells, but the activation of STAT3 induces differentiation of Th17 cells [31]. In this study, the number of Treg cells increased with CoQ10 treatment while Th17 differentiation was inhibited. There has been little information about the control of Th17 and Treg cells by CoQ10. It has been demonstrated that transcription of IL-17 is controlled by competitive binding of pSTAT3 and pSTAT5 [32]. Thus, pSTAT5 is a critical transcriptional factor for Foxp3 regulating differentiation of Treg cells. Our results revealed that while CoQ10 significantly induced pSTAT5+ T cells, pSTAT3+ T cells were reduced by CoQ10 treatment. It seems to be caused by the down-regulation of T cell transcriptional regulators such as ROR␥t, BATF and RUNX1 and the Q9 up-regulation of Foxp3. Hence, CoQ10 function to inhibit pSTAT3 and enhance pSTAT5 may be another efficient mechanism by which CoQ10 controls Th17/Treg balance. Oxidative stress is known to play a key role in RA pathogenesis. It has been reported that CoQ10 decreased iNOS and protected
Fig. 6. CoQ10 treatment reduces Th17 cell differentiation in CD4+ T cells isolated from SKG mice and CD4+ T cells isolated from human peripheral blood mononuclear cells. (A) Real-time reverse transcriptase-polymerase chain reaction of IL-17, CCL20, ROR␥t, BATF and RUNX1 mRNA in CD4+ T cells. (B) The production of TNF- and IL-21 induced by Th17 condition in the presence or absence of CoQ10. (C) After CoQ10 treatment, the populations of IL-17 and Foxp3 producing CD4+ T cells were analyzed by using antibodies specific for CD4, Foxp3 and IL-17 by intracellular flow cytometric analysis. (D) Real-time reverse transcriptase-polymerase chain reaction of IL-17, CCL20, RORc, Q15 and FoxP3 mRNA in CD4+ T cells. (E) The production of IL-17, -21 and TNF-␣ induced by Th17 condition in the presence or absence of CoQ10. (F) After several concentration of CoQ10 treatment, the populations of IL-17 and Foxp3 producing CD4+ T cells were analyzed by using antibodies specific for CD4, Foxp3 and IL-17 by intracellular flow cytometric analysis. Data are presented as the mean ± SD of three independent experiments (*P < 0.05, **P < 0.03, ***P < 0.001).
Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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endothelial cells from oxidative stress-induced injury [33]. More recently, CoQ10 acted as a suppressor of iNOS in the joints of osteoarthritis mice [34]. Our data show that the expression of iNOS was reduced after CoQ10 treatment in RA mice joints. As the expression of iNOS and nitrotyrosine indicates oxidative stress, down-regulating the expression of iNOS and nitrotyrosine in the joints of CoQ10 treated mice demonstrated a decrease in the oxidative stress compared to the vehicle treated mice. The inhibitor of Breg cells proliferation was reported as a suppressive factor of arthritis severity in RA murine model [35]. In this study, GCB cells expressing GL-7 are involved with the B
cell immune response and Tfh cells revealing ICOS and PD-1 are involved in the germinal center responses. GC B cells and Tfh cells produce memory B cells and long-lived plasma cells [36]. Our data showed that total IgG, IgG1 and IgG2a level reduced by CoQ10 may cause to inhibit differentiation of GCB cells and Tfh cells. These results suggest that the function of CoQ10 is involved in declining the B cell immune response. The reduction of osteoclastogenesis by CoQ10 treatment was revealed by both in vivo and in vitro. The number of TRAP+ multinucleated cells decreased in the joints of CoQ10 treated mice seems to have resulted in decreased expression of inflammatory cytokines
Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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that enhance osteoclastogenesis through the up-regulation of RANKL in the arthritic joint. It is well reported that the inhibition of osteoclastogenesis suppresses joint inflammation and severity of autoimmune arthritis [37]. Thus, CoQ10 can reveal therapeutic effect in autoimmune arthritis through inhibition of osteoclastogenesis. To our knowledge, the present study provides the first evidence that CoQ10 can regulate Th17 differentiation. By inhibiting STAT3 phosphorylation, CoQ10 concurrently suppresses Th17 and enhances Treg. Furthermore, it can be expected that CoQ10 may have therapeutic potential in other diseases where Th17 plays a major role in the pathogenesis of such as inflammatory bowel disease or graft-versus-host disease. Future research on this topic will be promising. There is little information in the investigation regarding control of Th17 cells by CoQ10. The present study suggests that CoQ10 can regulate Th17 differentiation by inhibiting pSTAT3 while enhancing differentiation of Treg cells via pSTAT5. The CoQ10 functions newly identified in this investigation indicate that it likely performs an important role in attenuating inflammation and may shed light on the pathogenesis of immune-mediated inflammatory diseases including RA. Moreover, CoQ10 can be expected to conduct a significant role in the selection of immune-mediated inflammatory therapeutic targets. This novel evidence suggests that CoQ10 might be a strong candidate for the treatment of RA.
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Competing interests
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The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgements
This study was supported by Research Fund of Seoul St. Mary’s Hospital, The Catholic University of Korea. This research was sup380 Q11 ported by the Bio & Medical Technology Development Program 381 of the National Research Foundation (NRF) funded by the Korean 382 government (MEST) (No. 2012M3A9C6049783). 383 Q10 379
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Please cite this article in press as: J.Y. Jhun, et al., Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice, Immunol Lett (2015), http://dx.doi.org/10.1016/j.imlet.2015.05.012
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