Effects of CKD-M808, a novel histone deacetylase 6 inhibitor, on fibroblast-like synoviocytes and regulatory T cells in rheumatoid arthritis

Abstract

Background: Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease whose hallmark is joint destruction caused by synovial proliferation and leukocyte infiltration in synovial membrane. Histone deacetylase (HDAC) inhibitor was suggested as a promising therapeutic agent in cancer and autoimmune diseases. Recently, the toxicity of pan-HDAC inhibitor (pan-HDACi) has been reported in clinical trials. HDAC6 is a member of HDAC family, mainly targeting non-histone proteins. We examined the therapeutic effect of selective inhibitor of HDAC6 in RA.

Objective: We investigated anti-inflammatory effects of a novel HDAC6 inhibitor, CKD-M808 (M808), on fibroblast-like synoviocytes (FLS), peripheral blood mononuclear cells (PBMCs) and regulatory T cells (Treg) in RA as well as adjuvant-induced arthritis (AIA) rat model.

Methods: Cell viability was analyzed by cell-counting kit 8 (CCK-8). RA FLS were incubated with HDAC6 inhibitor, M808 and tubastatin A as a positive control, and then stimulated with IL-1β. The levels of metalloproteinase (MMP) -1, MMP-3, IL-6, CCL2, CXCL8, and CXCL10 were measured in culture supernatants of FLS by Magnetic Luminex Screening Assay multiplex kit (Luminex assay) or enzyme-linked immunosorbent assay (ELISA). Expression of HDAC6, α-tubulin, acetylated tubulin, cortactin, acetylated cortactin, ICAM-1, VCAM-1, and GAPDH in FLS were assessed by western blot. Wound healing assay and cell adhesion assay in FLS were performed. RA PBMCs were incubated with HDAC6 inhibitors, tubastatin A or M808, and then stimulated with LPS. TNF-α, IL-1β, IL-6, and IL-10 were measured in culture supernatants of PBMCs by Luminex assay and ELISA. CD4+CD25- T cells were isolated from RA PBMCs and induced to regulatory T cells. The markers of induced Treg (iTreg) were analyzed by flow cytometry. CD4+CD25- T cells (effector T cells, Teff) from healthy donor or murine splenocytes were stained with carboxyfluorescein succinimidyl ester (CFSE) or eFluor®670 and cocultured with iTreg from RA patients or Treg from murine splenocytes respectively. The proliferation of healthy Teff was analyzed by flow cytometry. Adjuvant-induced arthritis (AIA) was developed in Lewis rat. Clinical score of arthritis was measured after M808 treatment.

Result: M808 decreased the production of MMP-1, MMP-3, IL-6, CCL2, CXCL8, and CXCL10 in RA FLS. M808 increased the acetylation of tubulin and cortactin and inhibited the expression of ICAM-1 and VCAM-1 in RA FLS. The migration of RA FLS and adhesion of U937 cells or Jurkat cells on RA FLS were declined with the treatment of M808. M808 reduced TNF-α and increased IL-10 production, but had negligible effects on IL-1β and IL-6 in RA PBMCs. The function of iTreg induced from RA cells under the presence of M808 significantly improved by suppressing the proliferation of Teff. M808 had enhanced suppressive function of Treg and improves the clinical score of arthritis in AIA rat model.

Conclusion: A novel HDAC6 inhibitor, M808 exhibits the suppressive effects on inflammation by reducing the production of MMPs, pro-inflammatory cytokines, and chemokines, decreasing cell migration and adhesion of RA FLS, and improving the suppressive function of Treg derived from RA patients. M808 improved clinical score of arthritis in AIA animal model. M808 could be a potential therapeutic agent in RA.