Therapeutic effect of a novel histone deacetylase 6 inhibitor, CKD-L, on collagen-induced arthritis in vivo and regulatory T cells in rheumatoid arthritis in vitro

Histone deacetylase (HDAC) and histone acetyltransferase (HAT) play important roles in the regulation of gene transcription [1]. The positively charged lysine in the N-terminal tail of histones is neutralized by acetylation of histone by HAT, and the binding affinity between the DNA backbone and histones is decreased because histones do not bind to the negatively charged phosphate groups in DNA. The decreased level of interaction between DNA and histones increases gene transcription by promoting the binding of transcription factors to DNA [2‐4].

In opposition to HAT, deacetylation of histone by HDAC represses gene transcription through chromatin condensation [5]. HDAC is classified into four classes based on DNA sequence similarity and function. Class I, II, and IV HDACs are classical HDACs that have a zinc-dependent active site, whereas class III HDACs are a family of nicotinamide adenine dinucleotide (NAD⁺)-dependent proteins. Class I HDACs (HDACs 1–3 and 8) are primarily found in the nucleus, and class II HDACs (HDAC 4–7, 9, and 10) are found in both the nucleus and the cytoplasm and can shuttle between the two. The class IV HDAC (HDAC11) shares structural similarities with both class I and class II HDACs. Class III HDACs (SirT 1–7) have distinct structures and different mechanisms of action. Their enzymatic activity depends on the cofactor NAD⁺ [1, 6, 7].

HDAC6 is primarily located in the cytoplasm and has a unique structure that contains two homologous catalytic domains and an ubiquitin binding domain at the C-terminal end [8, 9]. HDAC6 has been reported to involve many important biological processes, including cell migration, immune response, viral infection, and the degradation of misfolded proteins. The substrates of HDAC6 are α-tubulin, heat shock protein (HSP) 90, peroxiredoxin (PRX), and cortactin, and HDAC6 regulates acetylation of multiple proteins by forming various complexes with other partner proteins. These diverse functions of HDAC6 offer potential therapeutic targets in various diseases such as systemic lupus erythematosus, cancer, and diabetes [10, 11]. The inhibition of HDAC6 was reported to enhance the suppressive activity of regulatory T cells (Treg cells) in inflammation and autoimmunity [12].

Histone deacetylase inhibitors modulate the function of HDACs and activate and/or repress gene expression. HDAC inhibition leads to cell cycle arrest, cell growth, cell differentiation, and apoptotic death of transformed cells [13, 14]. HDAC inhibitors have been developed; the pan HDAC inhibitors, such as ITF 2357 and SAHA, inhibit all HDACs, and the selective HDAC inhibitors, such as Tubastatin A and Tubacin, inhibit HDAC6 specifically [15].

Epigenetic regulation plays an important role in inflammatory autoimmune diseases including rheumatoid arthritis (RA) through changes in histone modification [16]. RA is a chronic autoimmune disease characterized by inflammatory synovitis and progressive destruction of joint cartilage and bone, leading to swelling, pain, stiffness, and loss of function [17, 18]. The etiology of RA remains unclear, but genetic background and environmental factors play important roles in the disease [19].

Current medical treatments for RA include nonsteroidal anti-inflammatory drugs (NSAIDs) and disease-modifying antirheumatic drugs (DMARDs), including methotrexate (MTX), sulfasalazine, and tumor necrosis factor (TNF) inhibitors (infliximab, etanercept, and adalimumab) [20]. TNF is a proinflammatory cytokine that is associated with the development of RA. TNF blockade can be an effective treatment for RA [21]. However, about 40% of RA patients do not respond to antiTNF therapy, so novel therapies are required for patients who show no response [1, 5].

HDAC inhibitors have been reported to have potential therapeutic effects as anti-inflammatory agents in many studies including those on collagen-induced arthritis (CIA) [5, 22‐25]. HDAC inhibitors suppressed the production of interleukin (IL)-6 in RA fibroblast-like synoviocytes (FLS) and macrophages by promoting mRNA decay [26]. In RA, macrophages and T cells are major sources of proinflammatory cytokines [27]. The activation, survival, and apoptosis of macrophages are regulated by acetylation and deacetylation of histones. HDAC inhibitors suppress TNF and IL-6 production and cytokine gene transcription and induce apoptosis in macrophages [28]. HDAC inhibitors reduce the secretion of proinflammatory cytokines, such as TNF and IL-6, in peripheral blood mononuclear cells (PBMC) of RA patients and reduce the secretion of TNF, IL-1α, IL-1β, and interferon (IFN)-γ in lipopolysaccharide (LPS)-stimulated normal PBMC [1, 24]. Treg cells are a subset of CD4⁺ T cells that have an immunosuppressive role in immune tolerance [29]. Treg cells from RA patients are defective in suppressing proinflammatory cytokine production [30].

Tubastatin A inhibited HDAC6 and induced Foxp3 expression, resulting in enhanced Treg function [12]. In addition, Tubastatin A suppressed TNF and IL-6 from the THP1 macrophage cell line and inhibited CIA [31]. However, it is still unclear whether HDAC6-selective inhibitors have a similar immunoregulatory function in RA patients.

Here, we introduce a new HDAC6 selective inhibitor, CKD-L, developed by the Chong Kun Dang Pharmaceutical Corporation. Although the tertiary amine tricycle of Tubastatin A has excellent HDAC6-selective inhibitory activity, the poor PK profile and genotoxicity of Tubastatin A significantly limited its development for clinical use. CKD-L has a bicycle instead of a tertiary amine tricycle, resulting in better drug-like structure and profiles without any genotoxicity in the Ames test and the chromosome aberration test.

We investigated the therapeutic effect of the novel selective HDAC6 inhibitor, CKD-L, and compared its effect to those of the pan HDAC inhibitor, ITF 2357, and the selective HDAC6 inhibitor, Tubastatin A, in CIA, PBMC, and Treg cells from patients with RA. CKD-L showed comparable efficacy to Tubastatin A in the CIA model. Moreover, CKD-L showed immunomodulatory effect in RA patients, such as improvement of Treg function and suppression of TNF production in PBMC suggesting that HDAC6 may be an important therapeutic target protein to control RA, and that HDAC6-specific inhibitors such as CKD-L can be developed for clinical use.

We assessed the therapeutic effects of CKD-L on the severity of CIA in DBA1/J mice. After the onset of CIA, HDAC inhibitors were administered by subcutaneous injection. Arthritis progressed rapidly in the group treated with vehicle. CKD-L (30 mg/kg) significantly decreased the severity of arthritis compared with vehicle (p < 0.05), and Tubastatin A had a similar effect (Fig. 1a). The arthritis scores of the mice treated with 30 mg/kg CKD-L tended to be lower than those of the mice treated with 15 mg/kg CKD-L. Body weight was assessed every 2 days after the first drug administration. The body weights of the animals did not change significantly after administration of HDAC inhibitor (Fig. 1b). The changes in body weight between the first (day 21) and the last (day 38) assessment were not significantly different among the groups (Fig. 1c).

RA is characterized by synovial inflammation, bone erosion, cartilage damage, and leukocyte infiltration in the joints. To investigate the protective effects of CKD-L in the joints, the histological score was assessed in the knees and hind paws of mice by H&E staining. Significantly more synovial inflammation, bone erosion, cartilage damage, and leukocyte infiltration were observed in the knee joints and hind paws of the mice treated with vehicle (arrows in Fig. 2) compared to Tubastatin A or CKD-L (Fig. 2a). CKD-L effectively inhibited arthritis. Synovial inflammation (Fig. 2b), bone erosion (Fig. 2c), cartilage damage (Fig. 2d), and leukocyte infiltration (Fig. 2e) were significantly decreased in the groups treated with CKD-L or Tubastatin A compared to the vehicle-treated group. The histological score was calculated by summation of four parameters: synovial inflammation, bone erosion, cartilage damage, and leukocyte infiltration (Fig. 2f). The histological score was significantly lower in mice treated with CKD-L (p < 0.001) or Tubastatin A (p < 0.001). These findings suggest that CKD-L may be significantly effective for the treatment of CIA. There was no significant difference in histological score between Tubastatin A and CKD-L.

Treg cells have an immunosuppressive role [29]. It was reported that the suppressive function of Treg cells in RA patients is defective [30, 34]. We assessed the effect of CKD-L on Treg cells during their induction. CD4⁺CD25^– T cells were isolated from splenocytes of C57BL/6 mice. CD4⁺CD25^– T cells were incubated with vehicle or HDAC6 inhibitor (1 to 10 μM) in the presence of antiCD3/CD28 beads and recombinant TGF-β2 in a 48-well plate for 6 days. CTLA-4 expression in CD4⁺CD25⁺ Foxp3⁺ T cells was significantly increased after treatment with CKD-L (p < 0.001) or Tubastatin A (p < 0.05) compared to vehicle (Fig. 3).

Treg cells have an immunosuppressive role in immune tolerance [29]. It is known that the suppressive function of Treg cells from RA patients was defective in many studies [30, 34]. We assessed the effect of CKD-L on the function of Treg cells. Treg cells (CD4⁺CD25⁺ T cells) and Teff cells (CD4⁺CD25^– T cells) were isolated from splenocytes of C57BL/6 mice. Treg cells were mixed with eFluor®670-labeled Teff cells at ratios of 4:1 and 0:1. The mixed cells were incubated with vehicle or HDAC6 inhibitor (1 to 10 μM) in the present of antiCD3/CD28 beads for 4 days. Proliferation of Teff cells was inhibited after treatment with CKD-L and Tubastatin A in a dose-dependent manner compared to vehicle (Fig. 4a). As shown by suppression ratio (fold versus vehicle) at 1:4 ratio of Treg:Teff, the suppression ratio was increased 1.4 times by CKD-L treatment and 1.6 times by Tubastatin A treatment compared to vehicle treatment (p < 0.001, respectively) (Fig. 4b).

To investigate the effect of CKD-L on PBMC of RA patients, cell viability was determined using the MTT assay. Cell viability was not affected by high concentrations of CKD-L or Tubastatin A (Fig. 5). However, ITF 2357 decreased cell viability at concentrations above 1 μM. The HDAC inhibitors were used in the experiments at concentrations that would not affect cell viability.

Isolated PBMC from RA patients were cultured with LPS (100 ng/ml) and HDAC inhibitors at different concentrations (0.01 to 5 μM) for 24 h. The secretions of TNF, IL-10, and IL-1β in the cell culture supernatant were measured by multiplex immunoassay, and IL-6 secretion in the cell culture supernatant was measured by ELISA. CKD-L significantly inhibited TNF at concentrations of 0.01 μM and 5 μM (Fig. 6a). Also, CKD-L inhibited IL-1β at a concentration of 1 μM (Fig. 6b) and increased IL-10 at a concentration of 5 μM (Fig. 6d). ITF 2357 inhibited TNF at a concentration of 0.1 μM, and Tubastatin A inhibited TNF at concentrations of 1 μM and 5 μM. ITF 2357 and Tubastatin A had no effect on IL-1β and IL-10. IL-6 production did not change following treatment with any HDAC inhibitor (Fig. 6c).

Real-time PCR was conducted to measure the mRNA levels of TNF and IL-10. Total RNA was extracted from harvested cells and cDNA was synthesized by RT-PCR and then amplified. TNF mRNA expression was significantly decreased after treatment with a high concentration (5 μM) of CKD-L (p < 0.001) (Fig. 7). ITF 2357 and Tubastatin A also decreased TNF mRNA expression (p < 0.001). However, IL-10 mRNA levels did not change after treatment with CKD-L.

THP-1 cells were activated with PMA for 24 h to induce macrophage differentiation. After differentiation, they were treated with vehicle or HDAC inhibitor (0.1 to 10 μM) for 24 h and then treated with 100 ng/ml LPS for 4 h. TNF was significantly decreased after treatment with CKD-L in a dose-dependent manner (p < 0.001) (Fig. 8). TNF secretion was also significantly inhibited by ITF 2357 (p < 0.001) and Tubastatin A (p < 0.001).

Induced Treg cells derived from RA patients were mixed with CFSE-labeled Teff cells at ratios of 1:1, 0.3:1, and 0:1. The mixed cells were incubated with vehicle or HDAC inhibitor (0.01 to 5 μM) in the presence of Dynabeads® human T-activator CD3/C28 (one bead to four cells) for 3 days. Proliferation of Teff cells was inhibited after treatment with CKD-L (5 μM) and ITF 2357 (0.1 μM) compared to vehicle (Fig. 9a). As the proportion of induced Treg cells was increased, proliferation of Teff cells was inhibited. In addition, in the Teff cell-only condition, ITF 2357 inhibited the proliferation of Teff cells but CKD-L did not have an effect. Therefore, the suppressive effect of CKD-L seems to be mediated by Treg cells. Tubastatin A had no suppressive effect in the suppression assay. The suppression ratio (fold versus vehicle) at 1:1 ratio of Treg:Teff was increased 1.5 times by CKD-L treatment and 1.8 times by ITF 2357 treatment compared to vehicle treatment (p < 0.05 and p < 0.01, respectively) (Fig. 9b).

Epigenetic regulation potentially influences the pathogenesis of RA and can provide therapeutic targets for the treatment of RA [35]. HDAC inhibitors that modulate the activities of HDAC and HAT have been reported to have potential anti-inflammatory effects on RA in many studies [5, 22‐25]. In addition, HDAC inhibitors ameliorated joint inflammation and bone destruction in animal experiments, including in the CIA model [3, 5, 36]. Therefore, in the present study, we hypothesized that CKD-L could have beneficial effects on CIA. We found that CKD-L significantly decreased both the arthritis score and the histological score by blocking CIA progression.

We assessed the effect of CKD-L on the function of Treg cells. Treg cells and Teff cells were isolated from splenocytes of C57BL/6 mice and cocultured. Proliferation of Teff cells was inhibited after treatment with CKD-L or Tubastatin A in a dose-dependent manner. The suppression ratio (fold inhibition of cell proliferation by HDACi vs vehicle) was approximately two times greater after CKD-L treatment compared to vehicle treatment (data not shown).

In RA, activated CD4⁺ T cells have an important role in initiating and perpetuating chronic inflammation [37]. Based on their distinctive cytokine secretion profiles and functions, human CD4⁺ T cells can be divided into two major subtypes of cells, known as Th1 and Th2 cells. Th1 cells produce the proinflammatory cytokines IFN-γ, TNF, and IL-2, and promote macrophage activation, induce delayed-type hypersensitivity, and are involved in cell-mediated immunity. Th2 cells have been associated with downregulation of macrophage effector functions, they produce the anti-inflammatory cytokines IL-4, IL-5, IL-10, and IL-13, and mediate allergic immune responses [37‐39]. IgG2a production is associated with a Th1 response, whereas IgG1 production is associated with a Th2 response [40]. Therefore, we hypothesized that CKD-L can increase or maintain the level of IgG1 and decrease the level of IgG2a in serum from animals with CIA. We measured the levels of serum IgG1 and IgG2a by ELISA. However, the levels of serum IgG1 and IgG2a did not change significantly after CKD-L treatment (data not shown).

HDAC inhibitors have been reported to reduce the levels of TNF, IL-1α, IL-1β, and IFN-γ in LPS-stimulated normal PBMC and reduce the levels of proinflammatory cytokines such as TNF and IL-6 in PBMC of RA patients [1, 24, 26, 28]. It was also reported that inhibition of HDAC3 suppresses the inflammatory gene expression, including type I IFN production in RA FLS [41]. We found that CKD-L inhibited the secretion of TNF and IL-1β, and increased the secretion of IL-10 in PBMC of RA patients treated with LPS and HDAC inhibitors. Also, as assessed by real-time PCR, CKD-L significantly inhibited TNF mRNA levels, in accordance with the results of measurements taken from the cell culture supernatant. Tubastatin A and ITF 2357 inhibited TNF secretion, but had no effect on IL-1β or IL-10. CKD-L, which regulates several cytokines such as TNF, IL-1β, and IL-10, is expected to play an important role in abnormal immune responses in RA patients.

In RA, T cells and macrophages are major sources of proinflammatory cytokines and the activation, survival, and apoptosis of these cells may be regulated by HDAC and HAT [27]. It is known that HDAC inhibitors suppress TNF and IL-6 production and transcription of cytokines in macrophages, and induce apoptosis in macrophages [28]. We found that CKD-L inhibited TNF secretion in PMA-activated THP-1 in a dose-dependent manner. Increased histone acetylation can regulate TNF production by controlling the access of transcription factors to promoter motifs directly or by regulating nucleosome remodeling.

Treg cells play an immunosuppressive role by producing TGF-β or inhibiting Teff cells [29]. Treg cells from RA patients are defective in the suppression of proinflammatory cytokine production [30]. TNF in RA decreases the immunosuppressive effect by inducing the malfunction of Treg cells. It is known that inhibition of HDAC6 enhances the suppressive activity of Treg cells in inflammation and autoimmunity [12]. In the present study, in C57BL/6 mice, CKD-L increased CTLA-4 expression in CD4⁺CD25⁺Foxp3⁺ T cells more effectively than did Tubastatin A, and inhibited the proliferation of Teff cells on suppression assay. In the suppression assay on PBMC from RA patients, we found that CKD-L significantly inhibited the proliferation of Teff cells compared to vehicle. As the ratio of Treg cells increased, the proliferation of Teff cells was inhibited to a greater degree. Interestingly, ITF 2357 inhibited the proliferation of Teff cells when only Teff cells were present in culture. On the other hand, CKD-L had no effect when only Teff cells were present. Therefore, the suppressive effect of CKD-L seems to be regulated through Treg cells. Tubastatin A had no suppressive effect in the suppression assay. These results suggest that CKD-L promotes the suppressive function of Treg cells. HDAC inhibitors can increase acetylation of histone and Foxp3, leading to increased expression of Foxp3, and they increase the production and suppressive function of Treg cells [42]. Foxp3 proteins in Treg cells are present in a dynamic protein complex containing HAT and HDAC enzymes, and Foxp3 itself is acetylated on lysine residues. In addition, it has been reported that CTLA-4 expression is associated with acquisition of a suppressive function in activated human CD4⁺CD25^– T cells [43].

There are few studies on the comparative effect of selective versus nonselective HDAC inhibitors. HDAC6 has been reported to modulate many important biological processes, including cell migration, immune responses, viral infections, and the degradation of misfolded proteins. These diverse functions of HDAC6 can be used as potential therapeutic targets in various diseases, such as systemic lupus erythematosus, cancer, and diabetes [10, 11]. Our data demonstrated that CKD-L, a selective HDAC6 inhibitor, decreased the arthritis score and protected against joint destruction in the CIA model, and reduced the expressions of TNF and IL-1β, and increased the expression of IL-10 in PBMC from RA patients. Also, CKD-L increased CTLA-4 expression and the suppressive function of Treg cells. Our data suggest that CKD-L may have a beneficial effect in the treatment of RA and further studies are needed to establish its role as a potential therapeutic agent.

CKD-L, a selective HDAC6 inhibitor, decreased the arthritis score in CIA, reduced the expression of TNF and IL-1β, and increased the expression of IL-10 in PBMC from RA patients. CKD-L increased CTLA-4 expression and the suppressive function of Treg cells. These results suggest that CKD-L may have a beneficial effect in the treatment of rheumatoid arthritis.