International Journal of Rheumatic Diseases 2015; 18: 514–523
ORIGINAL ARTICLE
A novel histone deacetylase 6-selective inhibitor suppresses synovial inflammation and joint destruction in a collagen antibody-induced arthritis mouse model Jaejoon LEE,1 Eun Chung HONG,2 Hyemin JEONG,1 Ji Won HWANG,1 Hyungjin KIM,1 Eun-Kyung BAE,2 Joong Kyong AHN,3 Yoon-La CHOI,4 Jungho HAN,4 Hoon-Suk CHA1 and Eun-Mi KOH1 1 Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 2Samsung Biomedical Research Institute, 3Department of Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, and 4Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
Abstract Aim: To investigate the effects of Tubastatin A, a selective histone deacetylase-6 inhibitor, on synovial inflammation and joint destruction in a collagen antibody-induced arthritis (CAIA) mouse model. Methods: Collagen antibody-induced arthritis mice were given daily intraperitoneal injections of various concentrations of Tubastatin A (0, 10, 50, 100 mg/kg). The clinical score and paw thickness were measured. Mice were sacrificed on day 15, and the expression of tumor necrosis factor (TNF)-a, interleukin (IL)-1 and IL-6 in the serum were analyzed using enyme-linked immunosorbent assay (ELISA). Two pathologists independently measured the synovitis score. Micro-computed tomography (CT) scans of the joints were performed to quantify joint destruction. The expression of IL-6 from human fibroblast-like synoviocytes (FLSs) after incubation with various doses of Tubastatin A (0, 0.75, 1.5, 3 lmol/L) was measured using ELISA. Results: The clinical arthritis score was significantly attenuated and paw thickness was lower in the group treated with 100 mg/kg Tubastatin A compared with those treated with vehicle alone. The synovitis score was significantly reduced in the 100 mg/kg Tubastatin A-treated group compared with the control group. Micro-CT showed that quantitative measures of joint destruction were significantly attenuated in the 100 mg/kg Tubastatin A-treated group compared with the control. The expression of IL-6 in the sera was lower in the mice treated with Tubastatin A compared with the control. The expression of IL-6 in human FLSs decreased dose-dependently after incubation with Tubastatin A without affecting cell viability. Conclusions: Tubastatin A successfully ameliorated synovial inflammation and protected against joint destruction in CAIA mice, at least in part, by modulating IL-6 expression. Key words: rheumatoid arthritis, tubastatin, erosion, collagen antibody-induced arthritis.
INTRODUCTION Correspondence: Professor Eun-Mi Koh, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 135-710, Korea. Email: [email protected]
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by the proliferation and invasion of synovial tissues that leads to the destruction of bone and cartilage.1,2 Biological therapies have had a significant impact on the course of this disease and have prevented many disabilities that result from joint and
© 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
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cartilage destruction in many RA patients. Nevertheless, nonresponse rates among RA patients still remain at an unacceptable level. Therefore, there is an unmet need for innovative therapeutic interventions.3 A novel approach for the treatment of arthritis is the epigenetic modification of gene expression. Accumulating evidence suggests that epigenetic changes, including histone modifications, play an important role in inflammatory diseases such as RA.4,5 Histone deacetylases (HDACs) and histone acetyltransferases (HATs) induce post-translational modifications of the N-terminal tails of nuclear histone proteins and regulate gene transcription through remodeling of chromatin structure. Furthermore, HDACs and HATs are reported to have a wide range of non-histone targets, including modification of proteins important in transcriptional activation, nuclear shuffling and differentiation.6 The human genome encodes 18 different HDAC isoforms, which are grouped into four classes based on structural homology with HDACs found in yeast.7,8 Previous studies have shown that pan-HDAC inhibitors, such as trichostatin A, givinostat, suberoylanilide hydroxamic acid (SAHA) and MS-275, exhibit anti-arthritic effects in animal models of RA.9–12 These HDAC inhibitors are characterized by a pan-HDAC inhibitory profile that targets multiple HDAC isoforms, which may have undesirable side-effects such as fatigue, diarrhea, nausea, neutropenia and thrombocytopenia. Conversely, isoform-selective HDAC inhibitors may have reduced side-effects while retaining the ability to modulate targets. HDAC6 is a unique isoform because it acts primarily as a deacetylase for non-histone substrates, and this activity is consistent with non-epigenetic functions.13,14 The substrates for HDAC6 are cytoplasmic proteins such as a-tubulin, cortactin and heat shock protein 90 (Hsp90). Thus, HDAC6 plays an important role in regulating microtubule dynamics and microtubule-mediated processes, including cell–cell interactions, cell migration, metastasis and angiogenesis.15–17 Based on these functions, HDAC6 has become one of the most attractive targets for cancer treatment. Fibroblast-like synoviocytes (FLSs) derived from RA synovium are the primary effector cells that mediate cartilage destruction and exhibit unique features with aggressive and invasive properties that are reminiscent of cancer cells.18 Therefore, it is reasonable to hypothesize that HDAC6 inhibitors may be effective treatments for RA. Thus, the present study aimed to investigate the effect of Tubastatin A, a potent and selective HDAC6 inhibitor, on synovial inflammation and joint destruction in a collagen antibody-induced arthritis (CAIA) mouse
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model. We demonstrate that Tubastatin A ameliorates synovial inflammation and protects against joint destruction in CAIA mice. In addition, we show that Tubastatin A inhibits interleukin 6 (IL-6) production from human FLSs. Our findings strongly support the notion that selective HDAC6 inhibition may serve as a novel therapeutic target for the treatment of RA.
MATERIALS AND METHODS Animals, arthritis induction and Tubastatin A treatment Twenty-four 6-week-old male DBA/1J mice (Charles River Japan, Yokohama, Japan) were used to evaluate the anti-arthritic effect of Tubastatin A in vivo. All animals were treated in accordance with the guidelines and regulations of the Laboratory Animal Research Center at Sungkyunkwan University School of Medicine. Arthritis was induced using an arthritogenic cocktail of four monoclonal antibodies (mAbs) to type II collagen (Chondrex, Redmond, WA, USA) combined with lipopolysaccharide (LPS) simulation according to Terato’s method.19,20 The mice were injected intraperitoneally with 2 mg of mAb on days 0 and 1, followed by intraperitoneal (i.p.) injection of 50 mg of LPS on day 2. The treatment group (n = 18) was given a daily i.p. injection of Tubastatin A (Sigma-Aldrich, Oakville, ON, Canada) (10, 50 or 100 mg/kg of body weight, n = 6 in each group) until the end of the experiment on day 14 (Fig. 1a). Tubastatin A was dissolved in dimethyl sulfoxide (DMSO) and then diluted with phosphate-buffered saline (PBS) to the proper final concentration. Control mice (n = 6) were injected with 0.1% DMSO.
Clinical evaluation of arthritis Arthritis progression was monitored daily by recording paw thickness, body weight and clinical score of the mice. The clinical score was measured using a 4-point clinical scoring scale (0, no swelling or redness; 1, swelling/redness of the paw or one joint; 2, two joints involved; 3, more than two joints involved; and 4, severe arthritis of the entire paw and joints). The total score for clinical disease activity was the sum of the scores from all four paws (maximum score = 16). Paw thickness was measured with a vernier caliper. Two independent observers performed arthritis scoring and paw thickness measurements.
Histopathological examination Mice were anesthetized and euthanized on day 15. At this time, the paws and knee joints were removed
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for histopathological examination following routine fixation, decalcification and paraffin embedding of the tissues. The grading of the synovial membrane was carried out on routine hematoxylin and eosinstained slides. The samples were evaluated by two independent pathologists according to a validated synovitis score.21–23 Three features of chronic synovitis (hyperplasia of the lining cell layer, cellular density of the synovial stroma and inflammatory cell infiltration) were semi-quantitatively evaluated (from 0, absent to 3, strong), and each feature was separately graded.
ELISA for serum levels of inflammatory cytokines in mice Animals were anesthetized by isoflurane inhalation, and blood samples were obtained by heart puncture at the time of sacrifice. After collection of the whole blood, the blood was placed at room temperature for 20 min to allow it to clot. The clots were removed by centrifugation at 2000 9 g for 10 min in a refrigerated centrifuge. Serum was collected and stored at 70°C until further analysis was performed. The samples were assayed using a multiplex cytokine enzyme-linked immunosorbent assay (ELISA) kit (R & D system, Minneapolis, MN, USA) for IL-1b, IL-6 and tumor necrosis factor (TNF)-a. In addition, IL-1b, IL-6 and TNF-a were also assayed using the Fluorokine Mouse Multianalyte Profiling (MAP) Base Kit (R&D system, Minneapolis, MN, USA) according to the manufacturer’s instructions.
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Figure 1 Protocol for arthritis induction and the anti-arthritic effects of Tubastatin A. (a) Arthritis was induced as described in the Materials and Methods section, and a daily injection of Tubastatin A was given intraperitoneally from day 4 until day 14. (b) The clinical score was significantly attenuated in the 100 mg/kg-treated group compared with the control group beginning on day 10. (c) Paw thickness was significantly higher in the control group compared with the 100 mg/kg-treated group beginning on day 11. (d) All mice lost a small amount of weight, but no significant differences were observed between the groups. Data are expressed as the means standard error. **P < 0.05 and *P < 0.01 by Bonferroni multiple comparison test, control versus 100 mg/kgtreated group.
Micro CT imaging and 3D reconstruction Micro-computed tomography (CT) scanning was performed with the Small-Animal Imaging System (Inveon; Siemens Medical Solutions, Knoxville, TN, USA). First, the mice were sacrificed, and then the images were obtained. Next, the paws and knee joints were excised and fixed in 10% neutral buffered formalin. Image reconstructions were conducted using Inveon CT scanner software (Inveon Acquisition Workplace; IAW version 1.5, Siemens Medical Solutions, Knoxville, TN, USA). The voxel size was 40 lm, and the X-ray tube voltage was 70 kV with a current of 400 lA. An aluminium filter with a 1.5 mm thickness was used with 700 ms of exposure time. The X-ray projections were obtained at an angular sampling of 0.5° per projection for a full 360° scan. The dataset of the CT images was reconstructed using a filtered back projection algorithm with a Shepp-Logan filter. Using the sample reconstruction program, the bone volume, tissue volume and total bone surface area was measured.
Isolation and culture of human fibroblast-like synoviocytes The experimental protocol was approved by the Institutional Review Board of the Samsung Medical Center, and a signed consent form was obtained from each patient. Synovial tissue samples were obtained from RA patients at the time of arthroscopic synovectomy. Patients with RA were diagnosed according to the stan-
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dards of the American College of Rheumatology.24 The samples were minced, digested overnight with 5 mg/ mL type IV collagenase (Sigma, St. Louis, MO, USA) and 150 g/mL type I DNase (Sigma) and separated from undigested tissue by unit gravity sedimentation. After collecting the suspended cells in fresh tubes, the cells were harvested by centrifugation at 500 9 g for 10 min. The pellet was washed twice with Dulbecco’s modified Eagle’s media (DMEM; BioWhittaker Inc., Walkersville, MD, USA) containing 10% fetal bovine serum (FBS). The resuspended cells were plated at a concentration of 2 9 106/mL in a total volume of 1 mL/200 mm2 in a T-25 culture flask. After an overnight incubation, non-adherent cells were removed by replacing the culture medium, and the attached cells were cultured in DMEM supplemented with 10% FBS and 50 units/mL penicillin, 50 g/mL streptomycin and 0.025 g/mL amphotericin B until the cells reached 90% confluency. Primary cultured fibroblast-like synoviocytes (FLSs) were passaged three to four times over several weeks for the subsequent experiments.
ELISA to assess IL-6 levels in human FLS culture supernatants Rheumatoid arthritis FLS (n = 3) cells were plated in a six-well culture plate at 1 9 104 cells/well and were treated with various doses of Tubastatin A (0, 0.75, 1.5 or 3 lmol/L). The plates were incubated at 37°C in a humidified atmosphere. After 24 h, the cell culture supernatant in each well was collected and stored at 70°C until used for analysis. The levels of IL-6 in the culture supernatants were quantified using a commercially available ELISA kit according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). The plates were read at 450 nm using the xMark Microplate reader (Bio-Rad, Hercules, CA, USA). The concentration of IL-6 was calculated according to the standard curve.
Cell proliferation assay The survival and proliferation of RA FLS cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded at 1 9 103 cells/mL (n = 3) in triplicate in a 96-well
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Figure 2 Histological evaluation of arthritis. (a) Representative results demonstrating the histological appearance of the hind foot in the control and 100 mg/kg Tubastatin A-treated groups are shown. Synovial inflammation was considerably pronounced in the control group, while normal joint architecture was preserved in the 100 mg/kg Tubastatin A-treated group. (b–d) Two pathologists independently scored the severity of the synovial inflammation. Synovial hypertrophy, the density of resident cells and inflammatory cell infiltrates were significantly higher in the control group compared with the 100 mg/kg-treated group. *P < 0.01 Mann– Whitney test. The columns represent means standard error.
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microculture plate and were treated with different doses of Tubastatin A for 48 h (0, 0.75, 1.5 or 3 lmol/L). Cell Counting Kit-8 solution (Dojindo, Tokyo, Japan) was added for the final 4 h of the incubation, and absorbance at 450 nm was measured using the xMark Microplate reader (Bio-Rad).
Statistical analysis All analyses were performed using GraphPad Prism (version 4.0, GraphPad, San Diego, CA, USA). The statistical comparisons between the groups were analyzed using the Bonferroni/Dunn test and the Mann– Whitney U-test. P < 0.05 was considered statistically significant.
RESULTS Tubastatin A attenuated clinical arthritis in CAIA mice Collagen antibody-induced arthritis was induced as described in the Materials and Methods section, and daily injection of Tubastatin A was given intraperitoneally from day 4 until day 14 (Fig. 1a). Arthritis developed on day 4 in all CAIA mice, and the clinical score increased gradually until day 14 in the vehicle-treated mice. The clinical score in the Tubastatin A-treated mice was consistently less during the observation period
compared to the mice treated with vehicle. The clinical scores of mice treated with 100 mg/kg were significantly different from the control group beginning on day 10 (Fig. 1b). Paw thickness measurements showed a similar trend, and the difference between the 100 mg/ kg-treated group and the control group became statistically significant on day 11 (Fig. 1c). Daily body weight monitoring was used to assess toxicity. As shown in Fig. 1d, all mice lost a small amount of weight, but no significant differences were observed between the groups.
The histopathological score of synovial inflammation was reduced by Tubastatin A treatment Representative histopathological paw joint tissues were obtained from the control group and mice treated with 100 mg/kg Tubastatin A and are shown in Fig. 2a. Synovial inflammation was considerably pronounced in the control group compared to the well-preserved normal joint architecture observed in the 100 mg/kg Tubastatin A-treated group. Two pathologists measured the severity of the synovial inflammation. Synovial hypertrophy, the density of resident cells and inflammatory cell infiltrates were significantly higher in the control group compared with the 100 mg/kg Tubastatin A-treated group (Fig. 2b–d).
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Figure 3 The effect of Tubastatin A on cytokine production was measured using enzyme-linked immunosorbent assay (ELISA). (a) Mouse serum obtained on day 15 was used for analysis. Interleukin (IL)-6 expression decreased in the treatment group and was significantly lower in the 50 mg/kg-treated group compared with the control group. (b,c) No significant differences in IL-1 and tumor necrosis factor (TNF)-a expression were observed among the groups. *P < 0.05, Dunn’s multiple comparison test, control versus 50 mg/kg-treated group. The columns represent means standard error.
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Figure 4 The effect of Tubastatin A on joint destruction in CAIA. (a) Representative 3D reconstructed images of the hind foot demonstrate the protective effects of Tubastatin A. (b) Bone volume decreased in the control group compared with the treatment group. (c) Bone volume/tissue volume decreased significantly in the control group compared with 100 mg/kg Tubastatin A-treated group. (d) Bone surface area/bone volume was significantly higher in the control group compared with 100 mg/kg-treated group. *P < 0.05, Dunn’s multiple comparison test, control versus 100 mg/kg-treated group.
Tubastatin A reduces the expression of IL-6 in the serum of CAIA mice To evaluate the in vivo effects of Tubastatin A on inflammatory cytokine production, an ELISA was performed using mouse serum obtained on day 15. As shown in Figure 3a, IL-6 expression was inhibited in the treatment group and was significantly reduced in the 50 mg/kg group compared with the control group. However, no significant differences in IL-1 and TNF-a expression were observed among the groups (Fig. 3b,c).
Tubastatin A protects CAIA mice against joint destruction Next, we observed the effect of Tubastatin A on joint destruction in CAIA mice. Figure 4a shows a representative reconstructed 3D image of the left forefoot. Severe bone erosions were observed in the control mice, but the 100 mg/kg Tubastatin A-treated mice showed normal joint architecture. Bone volume was reduced in the
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control group compared with the treatment group (Fig. 4b), and bone volume/tissue volume was significantly reduced in the control group compared with the 100 mg/kg-treated group (Fig. 4c). In addition, bone surface area/bone volume, a quantitative measure of bony erosion, was significantly increased in the control group compared with the 100 mg/kg-treated group.
Tubastatin A reduces IL-6 expression in human FLSs Finally, the effect of Tubastatin A on IL-6 production by human FLSs was examined using ELISA. As shown in Figure 5a, IL-6 production by FLSs was inhibited in a dose-dependent manner following treatment with Tubastatin A. IL-6 production was significantly reduced when FLSs were treated with 3 lmol/L Tubastatin A compared with the control. An MTT assay was also performed and demonstrated that Tubastatin A inhibited IL-6 production without affecting cell viability.
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Figure 5 The effect of Tubastatin A on the interleukin (IL)-6 expression of human fibroblast-like synoviocytes (FLSs) was examined using enzyme-linked immunosorbent assay (ELISA). (a) The expression of IL-6 decreased in a dose-dependent manner following treatment with various concentrations of Tubastatin A. (b) An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that Tubastatin A inhibited IL-6 expression without affecting cell viability. *P < 0.05, Dunn’s multiple comparison test, control versus 3 lmol/L. The columns represent means standard error.
DISCUSSION The present study demonstrated that the HDAC6 selective inhibitor Tubastatin A can ameliorate synovial inflammation and protect against joint destruction in CAIA mice. HDAC inhibitors have emerged as potent anti-inflammatory agents in recent years25 and have been shown to inhibit the production of a range of cytokines in RA.4 Several studies have indicated that HDAC activities are dysregulated in RA. Kawabata et al. have demonstrated that nuclear HDAC activity was significantly higher in RA than in osteoarthritis and normal controls and correlated with the amount of cytoplasmic TNF-a.26 Stimulation with TNF-a significantly increased nuclear HDAC activity as well as HDAC1 messenger RNA (mRNA) and protein expression in RA FLSs. Similar results have been shown by Horiuchi et al.,27 which indicate that increased HDAC1 activity may play a role in RA pathogenesis. Specifically, they have shown that HDAC1 regulates the cell cycle in synovial tissue, thereby contributing to synovial inflammation. Based on these results, various HDAC inhibitors have been investigated in different animal models of arthritis, which have shown encouraging results, including reduced pro-inflammatory cytokine expression, disease severity and joint destruction.9–12 Most HDAC inhibitors that have been characterized to date display a pan-HDAC inhibitory profile. Given the multiple functions of HDAC isoenzymes and the pleiotropic effects of these pan-HDAC inhibitors, the mechanism of action and optimal strategies to exploit their therapeutic potential is not clearly defined. Moreover, the long-term safety of HDAC inhibitor use is a major concern because RA is a chronic disease that
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requires lifelong therapy.28,29 Clinical studies have indicated that the toxicity of anti-rheumatic drugs is one of the major reasons that patients switch between different therapies.29 Therefore, development of new generations of HDAC inhibitors with better safety profiles is required. Isoform selective HDAC inhibition may provide substantial advantages over broad-spectrum inhibition in terms of toxicity.13 However, despite the current understanding of the various HDAC family members, the HDAC isoenzyme responsible for the inflammatory processes of RA has yet to be identified. An interest in HDAC6 inhibitors has emerged in recent years because of its modulatory function in the acetylation of non-histone regulatory proteins implicated in cancer-related processes, including cell migration, metastasis and angiogenesis.30 FLSs derived from RA synovium are the primary effector cells that mediate cartilage destruction and exhibit unique features that are reminiscent of cancer-related processes.18 RA FLSs grow under anchorage-free conditions and can escape contact inhibition similarly to transformed cells.31 In addition, unlike FLSs from healthy individuals, RA FLSs co-implanted with human cartilage into severe combined immune-deficient mice spontaneously invade and destroy the human cartilage.32 These unique features of RA FLSs combined with the functions of HDAC6 strongly support the therapeutic use of HDAC6 inhibitors in the treatment of RA. HDAC6 modulates atubulin acetylation and therefore regulates microtubule network and cytoskeleton dynamics.33 Inhibition of HDAC6 may therefore block invasion of FLSs and prevent cartilage destruction. Hsp90 is another target of HDAC6, and the hyperacetylation of Hsp90 by HDAC6 inhibitors results in the depletion of oncogenic and
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antiapoptotic proteins, including epidermal growth factor receptor (EGFR), protein kinase B (Akt) and survivin.34 Hence, previously impaired apoptosis of FLSs may be restored. Additionally, the loss of Hsp90 function has been shown to inhibit the hypoxia-induced factor (HIF)-1a pathway, which has been implicated in the regulation of angiogenesis35 as well as the pathogenesis of RA.36 To date, there are few reports on the anti-arthritic effects of selective HDAC6 inhibitors. Vishwakarma et al. recently reported that Tubastatin showed a significant anti-arthritic effect in a collagen-induced arthritis model. Additionally, the expression of IL-6 in the paw tissue was significantly attenuated.37 Despite the use of different animal models for the experiment, our data are in close agreement with previous studies. Serum levels of IL-6 in our study were also lower in the treated group compared with the controls. However, serum levels of TNF-a and IL-1 were not significantly different among the groups. This result may be explained by the differential expression of inflammatory cytokines between the peripheral circulation and the target joint tissue. Expression of IL-6 in RA synovial tissue strongly correlates with disease activity and inflammation severity in RA,38 and targeting of the IL-6 receptor has been shown to have a strong clinical benefit in RA.39 Therefore, our study demonstrated that the anti-arthritic effect of the HDAC6 inhibitor is mediated, at least in part, by modulating IL-6 expression. Our study is novel in that we utilized 3D micro-CT to accurately quantify the effect of Tubastatin A on radiographic progression. To allow for a comparison of different-sized bone samples, we measured bone volume/ tissue volume. Bone surface area/bone volume was also measured to accurately reflect the loss of bone surface due to erosion. By utilizing this sophisticated technique, we confirmed that Tubastatin A protects against joint erosion. Moreover, we verified the results from the animal study using human FLSs in vitro. Interestingly, we found a dose-dependent decrease in IL-6 production in RA FLSs. These results correspond with the previously reported study by Grabiec et al.,40 which showed that IL-6 production from RA FLSs and macrophages is suppressed by HDAC inhibitors that act by accelerating mRNA decay. Inhibition of HDAC6 is not expected to produce severe toxicity because these compounds exhibited a high tolerability following preferential inhibition of this isoform.41,42 As expected, there was no significant difference in the body weight and the behavioral
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pattern of animals between the groups, and no deaths were observed.
CONCLUSION Our data demonstrated that Tubastatin A ameliorates synovial inflammation and protects against joint destruction in CAIA mice. Additionally, we found that the expression of IL-6 was reduced in human FLSs. These findings strongly support the notion that HDAC6 plays an important role in the pathogenesis of RA and may therefore serve as a novel therapeutic target in the treatment of RA.
ACKNOWLEDGEMENT This work was supported by a Samsung Biomedical Research Institute grant (SMR113091) and by the Basic Research Program of the National Research Foundation of Korea (NRF), Ministry of Education, Science and Technology (NRF-2013R1A1A1006243).
COMPETING INTERESTS The authors declare that they have no competing interests.
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37 Vishwakarma S, Iyer LR, Muley M et al. (2013) Tubastatin, a selective histone deacetylase 6 inhibitor shows antiinflammatory and anti-rheumatic effects. Int Immunopharmacol 16, 72–8. 38 Tak PP, Smeets TJ, Daha MR et al. (1997) Analysis of the synovial cell infiltrate in early rheumatoid synovial tissue in relation to local disease activity. Arthritis Rheum 40, 217–25. 39 Mima T, Nishimoto N (2009) Clinical value of blocking IL-6 receptor. Curr Opin Rheumatol 21, 224–30. 40 Grabiec AM, Korchynskyi O, Tak PP, Reedquist KA (2012) Histone deacetylase inhibitors suppress rheumatoid arthri-
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tis fibroblast-like synoviocyte and macrophage IL-6 production by accelerating mRNA decay. Ann Rheum Dis 71, 424–31. 41 Dallavalle S, Cincinelli R, Nannei R et al. (2009) Design, synthesis, and evaluation of biphenyl-4-yl-acrylohydroxamic acid derivatives as histone deacetylase (HDAC) inhibitors. Eur J Med Chem 44, 1900–12. 42 Santo L, Hideshima T, Kung AL et al. (2012) Preclinical activity, pharmacodynamic, and pharmacokinetic properties of a selective HDAC6 inhibitor, ACY-1215, in combination with bortezomib in multiple myeloma. Blood 119, 2579–89.
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ORIGINAL ARTICLE
A novel histone deacetylase 6-selective inhibitor suppresses synovial inflammation and joint destruction in a collagen antibody-induced arthritis mouse model Jaejoon LEE,1 Eun Chung HONG,2 Hyemin JEONG,1 Ji Won HWANG,1 Hyungjin KIM,1 Eun-Kyung BAE,2 Joong Kyong AHN,3 Yoon-La CHOI,4 Jungho HAN,4 Hoon-Suk CHA1 and Eun-Mi KOH1 1 Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 2Samsung Biomedical Research Institute, 3Department of Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, and 4Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
Abstract Aim: To investigate the effects of Tubastatin A, a selective histone deacetylase-6 inhibitor, on synovial inflammation and joint destruction in a collagen antibody-induced arthritis (CAIA) mouse model. Methods: Collagen antibody-induced arthritis mice were given daily intraperitoneal injections of various concentrations of Tubastatin A (0, 10, 50, 100 mg/kg). The clinical score and paw thickness were measured. Mice were sacrificed on day 15, and the expression of tumor necrosis factor (TNF)-a, interleukin (IL)-1 and IL-6 in the serum were analyzed using enyme-linked immunosorbent assay (ELISA). Two pathologists independently measured the synovitis score. Micro-computed tomography (CT) scans of the joints were performed to quantify joint destruction. The expression of IL-6 from human fibroblast-like synoviocytes (FLSs) after incubation with various doses of Tubastatin A (0, 0.75, 1.5, 3 lmol/L) was measured using ELISA. Results: The clinical arthritis score was significantly attenuated and paw thickness was lower in the group treated with 100 mg/kg Tubastatin A compared with those treated with vehicle alone. The synovitis score was significantly reduced in the 100 mg/kg Tubastatin A-treated group compared with the control group. Micro-CT showed that quantitative measures of joint destruction were significantly attenuated in the 100 mg/kg Tubastatin A-treated group compared with the control. The expression of IL-6 in the sera was lower in the mice treated with Tubastatin A compared with the control. The expression of IL-6 in human FLSs decreased dose-dependently after incubation with Tubastatin A without affecting cell viability. Conclusions: Tubastatin A successfully ameliorated synovial inflammation and protected against joint destruction in CAIA mice, at least in part, by modulating IL-6 expression. Key words: rheumatoid arthritis, tubastatin, erosion, collagen antibody-induced arthritis.
INTRODUCTION Correspondence: Professor Eun-Mi Koh, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 135-710, Korea. Email: [email protected]
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by the proliferation and invasion of synovial tissues that leads to the destruction of bone and cartilage.1,2 Biological therapies have had a significant impact on the course of this disease and have prevented many disabilities that result from joint and
© 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
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cartilage destruction in many RA patients. Nevertheless, nonresponse rates among RA patients still remain at an unacceptable level. Therefore, there is an unmet need for innovative therapeutic interventions.3 A novel approach for the treatment of arthritis is the epigenetic modification of gene expression. Accumulating evidence suggests that epigenetic changes, including histone modifications, play an important role in inflammatory diseases such as RA.4,5 Histone deacetylases (HDACs) and histone acetyltransferases (HATs) induce post-translational modifications of the N-terminal tails of nuclear histone proteins and regulate gene transcription through remodeling of chromatin structure. Furthermore, HDACs and HATs are reported to have a wide range of non-histone targets, including modification of proteins important in transcriptional activation, nuclear shuffling and differentiation.6 The human genome encodes 18 different HDAC isoforms, which are grouped into four classes based on structural homology with HDACs found in yeast.7,8 Previous studies have shown that pan-HDAC inhibitors, such as trichostatin A, givinostat, suberoylanilide hydroxamic acid (SAHA) and MS-275, exhibit anti-arthritic effects in animal models of RA.9–12 These HDAC inhibitors are characterized by a pan-HDAC inhibitory profile that targets multiple HDAC isoforms, which may have undesirable side-effects such as fatigue, diarrhea, nausea, neutropenia and thrombocytopenia. Conversely, isoform-selective HDAC inhibitors may have reduced side-effects while retaining the ability to modulate targets. HDAC6 is a unique isoform because it acts primarily as a deacetylase for non-histone substrates, and this activity is consistent with non-epigenetic functions.13,14 The substrates for HDAC6 are cytoplasmic proteins such as a-tubulin, cortactin and heat shock protein 90 (Hsp90). Thus, HDAC6 plays an important role in regulating microtubule dynamics and microtubule-mediated processes, including cell–cell interactions, cell migration, metastasis and angiogenesis.15–17 Based on these functions, HDAC6 has become one of the most attractive targets for cancer treatment. Fibroblast-like synoviocytes (FLSs) derived from RA synovium are the primary effector cells that mediate cartilage destruction and exhibit unique features with aggressive and invasive properties that are reminiscent of cancer cells.18 Therefore, it is reasonable to hypothesize that HDAC6 inhibitors may be effective treatments for RA. Thus, the present study aimed to investigate the effect of Tubastatin A, a potent and selective HDAC6 inhibitor, on synovial inflammation and joint destruction in a collagen antibody-induced arthritis (CAIA) mouse
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model. We demonstrate that Tubastatin A ameliorates synovial inflammation and protects against joint destruction in CAIA mice. In addition, we show that Tubastatin A inhibits interleukin 6 (IL-6) production from human FLSs. Our findings strongly support the notion that selective HDAC6 inhibition may serve as a novel therapeutic target for the treatment of RA.
MATERIALS AND METHODS Animals, arthritis induction and Tubastatin A treatment Twenty-four 6-week-old male DBA/1J mice (Charles River Japan, Yokohama, Japan) were used to evaluate the anti-arthritic effect of Tubastatin A in vivo. All animals were treated in accordance with the guidelines and regulations of the Laboratory Animal Research Center at Sungkyunkwan University School of Medicine. Arthritis was induced using an arthritogenic cocktail of four monoclonal antibodies (mAbs) to type II collagen (Chondrex, Redmond, WA, USA) combined with lipopolysaccharide (LPS) simulation according to Terato’s method.19,20 The mice were injected intraperitoneally with 2 mg of mAb on days 0 and 1, followed by intraperitoneal (i.p.) injection of 50 mg of LPS on day 2. The treatment group (n = 18) was given a daily i.p. injection of Tubastatin A (Sigma-Aldrich, Oakville, ON, Canada) (10, 50 or 100 mg/kg of body weight, n = 6 in each group) until the end of the experiment on day 14 (Fig. 1a). Tubastatin A was dissolved in dimethyl sulfoxide (DMSO) and then diluted with phosphate-buffered saline (PBS) to the proper final concentration. Control mice (n = 6) were injected with 0.1% DMSO.
Clinical evaluation of arthritis Arthritis progression was monitored daily by recording paw thickness, body weight and clinical score of the mice. The clinical score was measured using a 4-point clinical scoring scale (0, no swelling or redness; 1, swelling/redness of the paw or one joint; 2, two joints involved; 3, more than two joints involved; and 4, severe arthritis of the entire paw and joints). The total score for clinical disease activity was the sum of the scores from all four paws (maximum score = 16). Paw thickness was measured with a vernier caliper. Two independent observers performed arthritis scoring and paw thickness measurements.
Histopathological examination Mice were anesthetized and euthanized on day 15. At this time, the paws and knee joints were removed
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for histopathological examination following routine fixation, decalcification and paraffin embedding of the tissues. The grading of the synovial membrane was carried out on routine hematoxylin and eosinstained slides. The samples were evaluated by two independent pathologists according to a validated synovitis score.21–23 Three features of chronic synovitis (hyperplasia of the lining cell layer, cellular density of the synovial stroma and inflammatory cell infiltration) were semi-quantitatively evaluated (from 0, absent to 3, strong), and each feature was separately graded.
ELISA for serum levels of inflammatory cytokines in mice Animals were anesthetized by isoflurane inhalation, and blood samples were obtained by heart puncture at the time of sacrifice. After collection of the whole blood, the blood was placed at room temperature for 20 min to allow it to clot. The clots were removed by centrifugation at 2000 9 g for 10 min in a refrigerated centrifuge. Serum was collected and stored at 70°C until further analysis was performed. The samples were assayed using a multiplex cytokine enzyme-linked immunosorbent assay (ELISA) kit (R & D system, Minneapolis, MN, USA) for IL-1b, IL-6 and tumor necrosis factor (TNF)-a. In addition, IL-1b, IL-6 and TNF-a were also assayed using the Fluorokine Mouse Multianalyte Profiling (MAP) Base Kit (R&D system, Minneapolis, MN, USA) according to the manufacturer’s instructions.
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Figure 1 Protocol for arthritis induction and the anti-arthritic effects of Tubastatin A. (a) Arthritis was induced as described in the Materials and Methods section, and a daily injection of Tubastatin A was given intraperitoneally from day 4 until day 14. (b) The clinical score was significantly attenuated in the 100 mg/kg-treated group compared with the control group beginning on day 10. (c) Paw thickness was significantly higher in the control group compared with the 100 mg/kg-treated group beginning on day 11. (d) All mice lost a small amount of weight, but no significant differences were observed between the groups. Data are expressed as the means standard error. **P < 0.05 and *P < 0.01 by Bonferroni multiple comparison test, control versus 100 mg/kgtreated group.
Micro CT imaging and 3D reconstruction Micro-computed tomography (CT) scanning was performed with the Small-Animal Imaging System (Inveon; Siemens Medical Solutions, Knoxville, TN, USA). First, the mice were sacrificed, and then the images were obtained. Next, the paws and knee joints were excised and fixed in 10% neutral buffered formalin. Image reconstructions were conducted using Inveon CT scanner software (Inveon Acquisition Workplace; IAW version 1.5, Siemens Medical Solutions, Knoxville, TN, USA). The voxel size was 40 lm, and the X-ray tube voltage was 70 kV with a current of 400 lA. An aluminium filter with a 1.5 mm thickness was used with 700 ms of exposure time. The X-ray projections were obtained at an angular sampling of 0.5° per projection for a full 360° scan. The dataset of the CT images was reconstructed using a filtered back projection algorithm with a Shepp-Logan filter. Using the sample reconstruction program, the bone volume, tissue volume and total bone surface area was measured.
Isolation and culture of human fibroblast-like synoviocytes The experimental protocol was approved by the Institutional Review Board of the Samsung Medical Center, and a signed consent form was obtained from each patient. Synovial tissue samples were obtained from RA patients at the time of arthroscopic synovectomy. Patients with RA were diagnosed according to the stan-
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dards of the American College of Rheumatology.24 The samples were minced, digested overnight with 5 mg/ mL type IV collagenase (Sigma, St. Louis, MO, USA) and 150 g/mL type I DNase (Sigma) and separated from undigested tissue by unit gravity sedimentation. After collecting the suspended cells in fresh tubes, the cells were harvested by centrifugation at 500 9 g for 10 min. The pellet was washed twice with Dulbecco’s modified Eagle’s media (DMEM; BioWhittaker Inc., Walkersville, MD, USA) containing 10% fetal bovine serum (FBS). The resuspended cells were plated at a concentration of 2 9 106/mL in a total volume of 1 mL/200 mm2 in a T-25 culture flask. After an overnight incubation, non-adherent cells were removed by replacing the culture medium, and the attached cells were cultured in DMEM supplemented with 10% FBS and 50 units/mL penicillin, 50 g/mL streptomycin and 0.025 g/mL amphotericin B until the cells reached 90% confluency. Primary cultured fibroblast-like synoviocytes (FLSs) were passaged three to four times over several weeks for the subsequent experiments.
ELISA to assess IL-6 levels in human FLS culture supernatants Rheumatoid arthritis FLS (n = 3) cells were plated in a six-well culture plate at 1 9 104 cells/well and were treated with various doses of Tubastatin A (0, 0.75, 1.5 or 3 lmol/L). The plates were incubated at 37°C in a humidified atmosphere. After 24 h, the cell culture supernatant in each well was collected and stored at 70°C until used for analysis. The levels of IL-6 in the culture supernatants were quantified using a commercially available ELISA kit according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). The plates were read at 450 nm using the xMark Microplate reader (Bio-Rad, Hercules, CA, USA). The concentration of IL-6 was calculated according to the standard curve.
Cell proliferation assay The survival and proliferation of RA FLS cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded at 1 9 103 cells/mL (n = 3) in triplicate in a 96-well
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Figure 2 Histological evaluation of arthritis. (a) Representative results demonstrating the histological appearance of the hind foot in the control and 100 mg/kg Tubastatin A-treated groups are shown. Synovial inflammation was considerably pronounced in the control group, while normal joint architecture was preserved in the 100 mg/kg Tubastatin A-treated group. (b–d) Two pathologists independently scored the severity of the synovial inflammation. Synovial hypertrophy, the density of resident cells and inflammatory cell infiltrates were significantly higher in the control group compared with the 100 mg/kg-treated group. *P < 0.01 Mann– Whitney test. The columns represent means standard error.
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microculture plate and were treated with different doses of Tubastatin A for 48 h (0, 0.75, 1.5 or 3 lmol/L). Cell Counting Kit-8 solution (Dojindo, Tokyo, Japan) was added for the final 4 h of the incubation, and absorbance at 450 nm was measured using the xMark Microplate reader (Bio-Rad).
Statistical analysis All analyses were performed using GraphPad Prism (version 4.0, GraphPad, San Diego, CA, USA). The statistical comparisons between the groups were analyzed using the Bonferroni/Dunn test and the Mann– Whitney U-test. P < 0.05 was considered statistically significant.
RESULTS Tubastatin A attenuated clinical arthritis in CAIA mice Collagen antibody-induced arthritis was induced as described in the Materials and Methods section, and daily injection of Tubastatin A was given intraperitoneally from day 4 until day 14 (Fig. 1a). Arthritis developed on day 4 in all CAIA mice, and the clinical score increased gradually until day 14 in the vehicle-treated mice. The clinical score in the Tubastatin A-treated mice was consistently less during the observation period
compared to the mice treated with vehicle. The clinical scores of mice treated with 100 mg/kg were significantly different from the control group beginning on day 10 (Fig. 1b). Paw thickness measurements showed a similar trend, and the difference between the 100 mg/ kg-treated group and the control group became statistically significant on day 11 (Fig. 1c). Daily body weight monitoring was used to assess toxicity. As shown in Fig. 1d, all mice lost a small amount of weight, but no significant differences were observed between the groups.
The histopathological score of synovial inflammation was reduced by Tubastatin A treatment Representative histopathological paw joint tissues were obtained from the control group and mice treated with 100 mg/kg Tubastatin A and are shown in Fig. 2a. Synovial inflammation was considerably pronounced in the control group compared to the well-preserved normal joint architecture observed in the 100 mg/kg Tubastatin A-treated group. Two pathologists measured the severity of the synovial inflammation. Synovial hypertrophy, the density of resident cells and inflammatory cell infiltrates were significantly higher in the control group compared with the 100 mg/kg Tubastatin A-treated group (Fig. 2b–d).
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Figure 3 The effect of Tubastatin A on cytokine production was measured using enzyme-linked immunosorbent assay (ELISA). (a) Mouse serum obtained on day 15 was used for analysis. Interleukin (IL)-6 expression decreased in the treatment group and was significantly lower in the 50 mg/kg-treated group compared with the control group. (b,c) No significant differences in IL-1 and tumor necrosis factor (TNF)-a expression were observed among the groups. *P < 0.05, Dunn’s multiple comparison test, control versus 50 mg/kg-treated group. The columns represent means standard error.
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Figure 4 The effect of Tubastatin A on joint destruction in CAIA. (a) Representative 3D reconstructed images of the hind foot demonstrate the protective effects of Tubastatin A. (b) Bone volume decreased in the control group compared with the treatment group. (c) Bone volume/tissue volume decreased significantly in the control group compared with 100 mg/kg Tubastatin A-treated group. (d) Bone surface area/bone volume was significantly higher in the control group compared with 100 mg/kg-treated group. *P < 0.05, Dunn’s multiple comparison test, control versus 100 mg/kg-treated group.
Tubastatin A reduces the expression of IL-6 in the serum of CAIA mice To evaluate the in vivo effects of Tubastatin A on inflammatory cytokine production, an ELISA was performed using mouse serum obtained on day 15. As shown in Figure 3a, IL-6 expression was inhibited in the treatment group and was significantly reduced in the 50 mg/kg group compared with the control group. However, no significant differences in IL-1 and TNF-a expression were observed among the groups (Fig. 3b,c).
Tubastatin A protects CAIA mice against joint destruction Next, we observed the effect of Tubastatin A on joint destruction in CAIA mice. Figure 4a shows a representative reconstructed 3D image of the left forefoot. Severe bone erosions were observed in the control mice, but the 100 mg/kg Tubastatin A-treated mice showed normal joint architecture. Bone volume was reduced in the
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control group compared with the treatment group (Fig. 4b), and bone volume/tissue volume was significantly reduced in the control group compared with the 100 mg/kg-treated group (Fig. 4c). In addition, bone surface area/bone volume, a quantitative measure of bony erosion, was significantly increased in the control group compared with the 100 mg/kg-treated group.
Tubastatin A reduces IL-6 expression in human FLSs Finally, the effect of Tubastatin A on IL-6 production by human FLSs was examined using ELISA. As shown in Figure 5a, IL-6 production by FLSs was inhibited in a dose-dependent manner following treatment with Tubastatin A. IL-6 production was significantly reduced when FLSs were treated with 3 lmol/L Tubastatin A compared with the control. An MTT assay was also performed and demonstrated that Tubastatin A inhibited IL-6 production without affecting cell viability.
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Figure 5 The effect of Tubastatin A on the interleukin (IL)-6 expression of human fibroblast-like synoviocytes (FLSs) was examined using enzyme-linked immunosorbent assay (ELISA). (a) The expression of IL-6 decreased in a dose-dependent manner following treatment with various concentrations of Tubastatin A. (b) An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that Tubastatin A inhibited IL-6 expression without affecting cell viability. *P < 0.05, Dunn’s multiple comparison test, control versus 3 lmol/L. The columns represent means standard error.
DISCUSSION The present study demonstrated that the HDAC6 selective inhibitor Tubastatin A can ameliorate synovial inflammation and protect against joint destruction in CAIA mice. HDAC inhibitors have emerged as potent anti-inflammatory agents in recent years25 and have been shown to inhibit the production of a range of cytokines in RA.4 Several studies have indicated that HDAC activities are dysregulated in RA. Kawabata et al. have demonstrated that nuclear HDAC activity was significantly higher in RA than in osteoarthritis and normal controls and correlated with the amount of cytoplasmic TNF-a.26 Stimulation with TNF-a significantly increased nuclear HDAC activity as well as HDAC1 messenger RNA (mRNA) and protein expression in RA FLSs. Similar results have been shown by Horiuchi et al.,27 which indicate that increased HDAC1 activity may play a role in RA pathogenesis. Specifically, they have shown that HDAC1 regulates the cell cycle in synovial tissue, thereby contributing to synovial inflammation. Based on these results, various HDAC inhibitors have been investigated in different animal models of arthritis, which have shown encouraging results, including reduced pro-inflammatory cytokine expression, disease severity and joint destruction.9–12 Most HDAC inhibitors that have been characterized to date display a pan-HDAC inhibitory profile. Given the multiple functions of HDAC isoenzymes and the pleiotropic effects of these pan-HDAC inhibitors, the mechanism of action and optimal strategies to exploit their therapeutic potential is not clearly defined. Moreover, the long-term safety of HDAC inhibitor use is a major concern because RA is a chronic disease that
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requires lifelong therapy.28,29 Clinical studies have indicated that the toxicity of anti-rheumatic drugs is one of the major reasons that patients switch between different therapies.29 Therefore, development of new generations of HDAC inhibitors with better safety profiles is required. Isoform selective HDAC inhibition may provide substantial advantages over broad-spectrum inhibition in terms of toxicity.13 However, despite the current understanding of the various HDAC family members, the HDAC isoenzyme responsible for the inflammatory processes of RA has yet to be identified. An interest in HDAC6 inhibitors has emerged in recent years because of its modulatory function in the acetylation of non-histone regulatory proteins implicated in cancer-related processes, including cell migration, metastasis and angiogenesis.30 FLSs derived from RA synovium are the primary effector cells that mediate cartilage destruction and exhibit unique features that are reminiscent of cancer-related processes.18 RA FLSs grow under anchorage-free conditions and can escape contact inhibition similarly to transformed cells.31 In addition, unlike FLSs from healthy individuals, RA FLSs co-implanted with human cartilage into severe combined immune-deficient mice spontaneously invade and destroy the human cartilage.32 These unique features of RA FLSs combined with the functions of HDAC6 strongly support the therapeutic use of HDAC6 inhibitors in the treatment of RA. HDAC6 modulates atubulin acetylation and therefore regulates microtubule network and cytoskeleton dynamics.33 Inhibition of HDAC6 may therefore block invasion of FLSs and prevent cartilage destruction. Hsp90 is another target of HDAC6, and the hyperacetylation of Hsp90 by HDAC6 inhibitors results in the depletion of oncogenic and
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antiapoptotic proteins, including epidermal growth factor receptor (EGFR), protein kinase B (Akt) and survivin.34 Hence, previously impaired apoptosis of FLSs may be restored. Additionally, the loss of Hsp90 function has been shown to inhibit the hypoxia-induced factor (HIF)-1a pathway, which has been implicated in the regulation of angiogenesis35 as well as the pathogenesis of RA.36 To date, there are few reports on the anti-arthritic effects of selective HDAC6 inhibitors. Vishwakarma et al. recently reported that Tubastatin showed a significant anti-arthritic effect in a collagen-induced arthritis model. Additionally, the expression of IL-6 in the paw tissue was significantly attenuated.37 Despite the use of different animal models for the experiment, our data are in close agreement with previous studies. Serum levels of IL-6 in our study were also lower in the treated group compared with the controls. However, serum levels of TNF-a and IL-1 were not significantly different among the groups. This result may be explained by the differential expression of inflammatory cytokines between the peripheral circulation and the target joint tissue. Expression of IL-6 in RA synovial tissue strongly correlates with disease activity and inflammation severity in RA,38 and targeting of the IL-6 receptor has been shown to have a strong clinical benefit in RA.39 Therefore, our study demonstrated that the anti-arthritic effect of the HDAC6 inhibitor is mediated, at least in part, by modulating IL-6 expression. Our study is novel in that we utilized 3D micro-CT to accurately quantify the effect of Tubastatin A on radiographic progression. To allow for a comparison of different-sized bone samples, we measured bone volume/ tissue volume. Bone surface area/bone volume was also measured to accurately reflect the loss of bone surface due to erosion. By utilizing this sophisticated technique, we confirmed that Tubastatin A protects against joint erosion. Moreover, we verified the results from the animal study using human FLSs in vitro. Interestingly, we found a dose-dependent decrease in IL-6 production in RA FLSs. These results correspond with the previously reported study by Grabiec et al.,40 which showed that IL-6 production from RA FLSs and macrophages is suppressed by HDAC inhibitors that act by accelerating mRNA decay. Inhibition of HDAC6 is not expected to produce severe toxicity because these compounds exhibited a high tolerability following preferential inhibition of this isoform.41,42 As expected, there was no significant difference in the body weight and the behavioral
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pattern of animals between the groups, and no deaths were observed.
CONCLUSION Our data demonstrated that Tubastatin A ameliorates synovial inflammation and protects against joint destruction in CAIA mice. Additionally, we found that the expression of IL-6 was reduced in human FLSs. These findings strongly support the notion that HDAC6 plays an important role in the pathogenesis of RA and may therefore serve as a novel therapeutic target in the treatment of RA.
ACKNOWLEDGEMENT This work was supported by a Samsung Biomedical Research Institute grant (SMR113091) and by the Basic Research Program of the National Research Foundation of Korea (NRF), Ministry of Education, Science and Technology (NRF-2013R1A1A1006243).
COMPETING INTERESTS The authors declare that they have no competing interests.
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