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

Abstract

Background: Epigenetic regulation by histone deacetylase (HDAC) and histone acetyltransferase (HAT) plays an important role in several types of inflammatory arthritis, including rheumatoid arthritis (RA). RA is a chronic autoimmune disease characterized by inflammatory synovitis and progressive destruction of joint cartilage and bone leading to pain, swelling, and loss of function. HDAC inhibitor has recently been reported to have a therapeutic effect as an anti-inflammatory agent in collagen-induced arthritis (CIA).

Objective: We investigated the therapeutic effect of a new selective HDAC6 inhibitor, CKD-L, compared to ITF 2357 or Tubastatin A on CIA and regulatory T (Treg) cells in patients with RA.

Methods: CIA was induced by bovine type II collagen (CII) in DBA/1J mice. Mice were treated with vehicle (n=10), CKD-L (15 or 30 mg/kg, n=10, respectively), or Tubastatin A (30 mg/kg, n=9) by subcutaneous injection every day for 18 days. Arthritis score was assessed twice weekly after the onset of arthritis. Histological analysis was performed by H&E stain. CD4+CD25- T cells were isolated from splenocytes of naive C57BL/6 mice and incubated with anti-CD3/CD28 beads, TGF-β and HDAC6 inhibitor (1 to 10 μM) for 6 days. Cytotoxic T lymphocyte associated protein 4 (CTLA-4) expression in induced Treg cells was analyzed by flow cytometry. RA PBMCs were cultured in the presence of 100 ng/ml lipopolysaccharide with or without HDAC inhibitor (CKD-L and Tubastatin A, 0.01 to 5 μM, ITF 2357, 0.01 to 0.1 μM) for 24 hours. The secretions of TNF-α, IL-10, IL-1β and IL-6 in the culture supernatant were measured by ELISA. TNF-α and IL-10 mRNA expression levels were analyzed by real-time PCR. THP-1 cells were activated with PMA for 24 hours to induce macrophage differentiation. PMA-activated THP-1 was treated with vehicle or HDAC inhibitor (0.1 to 10 μM) for 24 hours and then with 100 ng/ml of LPS for 4 hours. The secretion of TNF-α was measured by ELISA. CD4+CD25- T cells from RA patients were cultured with anti-CD3 antibody, anti-CD28 antibody, IL-2, TGF-β, and 1,25-dihydroxyvitamin D3 for 5 days. Induced Treg cells were incubated for 3 days with 5 μM carboxyfluorescein succinmidyl ester (CFSE)-Teff cells in the presence of anti-CD3/CD28 beads and HDAC inhibitor (ITF 2357, 0.01 to 0.1 μM, CKD-L and Tubastatin A, 0.01 to 5 μM). The proliferation of Teff cells was analyzed by flow cytometry.

Result: In the CIA model, CKD-L and Tubastatin A significantly decreased arthritis score (CKD-L, p < 0.05

Tubastatin A, p < 0.01) and histological score (CKD-L and Tubastatin A, both p < 0.001). CTLA-4 expression in Foxp3+ T cells was significantly increased after treatment with CKD-L (p < 0.001) and Tubastatin A (p < 0.05). In RA PBMC, CKD-L significantly inhibited TNF-α and IL-1β, and increased IL-10. ITF 2357 and Tubastatin A inhibited TNF-α, but had no effect on IL-1β or IL-10. IL-6 was not altered by treatment with any HDAC inhibitor. CKD-L (p < 0.01), ITF 2357 (p < 0.001), and Tubastatin A (p < 0.001) significantly inhibited TNF-α mRNA expression. TNF-α secretion from PMA-activated THP-1 cells was reduced after treatment with CKD-L (p < 0.001) and Tubastatin A (p < 0.001). When induced Treg cells and Teff cells, the proliferation of Teff cells was significantly inhibited after treatment with CKD-L 5 μM (p < 0.05) and ITF 2357 0.1 μM (p < 0.001) compared to vehicle. As the proportion of induced Treg cells increased, Teff cell proliferation was inhibited to a greater degree. Tubastatin A had no effect on inhibition of proliferation.

Conclusion: CKD-L, a selective HDAC6 inhibitor, decreased arthritis score in CIA, reduced the expression of TNF-α 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.