IL-12/IL-23p40 identified as a downstream target of apremilast in ex vivo models of arthritis

Abstract

Background: Apremilast (Otezla^®) is a phosphodiesterase 4 (PDE4) inhibitor approved for the treatment of psoriasis and psoriatic arthritis (PsA), but the reason why apremilast shows clinical effect is not fully understood. The objective of this study was to study the downstream effects of apremilast on cells of inflamed joints in immune-mediated inflammatory arthritis.

Methods: Synovial fluid was obtained from patients with active rheumatoid arthritis (RA), PsA or peripheral spondyloarthritis (SpA; n = 18). The in vitro models consisted of synovial fluid mononuclear cells (SFMCs) or fibroblast-like synovial cells (FLSs) cultured for 48 h, SFMCs cultured for 21 days, an osteoclast pit formation assay, and a mineralization assay.

Results: In SFMCs cultured for 48 h, apremilast decreased the production of interleukin (IL)-12/IL-23p40 (the shared subunit of IL-12 and IL-23), colony-stimulating factor 1, CD6, and CD40 and increased the production of C-X-C motif chemokine 5 dose-dependently. Apremilast had a very different response signature compared with the tumor necrosis factor alpha inhibitor adalimumab with a substantially greater inhibition of IL-12/IL-23p40. In SFMCs cultured for 21 days, apremilast increased the secretion of IL-10. In FLS cultures, apremilast decreased matrix metalloproteinase-3 production. Apremilast decreased osteoclastogenesis but did not affect mineralization by human osteoblasts.

Conclusion: This study reveals the downstream effects of apremilast in ex vivo models of arthritis with a strong inhibition of IL-12/IL-23p40 by SFMCs. Our findings could explain some of the efficacy of apremilast seen in IL-12/IL-23-driven immune-mediated inflammatory diseases such as psoriasis and PsA.

Keywords: PALACE; apremilast; cyclic adenosine monophosphate; phosphodiesterase 4; psoriatic arthritis; rheumatoid arthritis; spondyloarthritis.

Figure 1.

(a) Secretion of IL-12/IL-23p40 (n = 9) and IL-8 (n = 11) by SFMCs cultured for 48 h untreated (UT) or treated with DMSO control, apremilast or ADA. Data were presented as NPX values, which is an arbitrary unit on log₂ scale. A decrease in one NPX corresponds to a two-fold decrease of the concentration. (b) Secretion of IL-10 (n = 14) and MCP-1 (n = 14) by SFMCs cultured for 21 days untreated (UT) or treated with DMSO control, apremilast or ADA. Data were normalized to untreated cultures and expressed as a ratio. (c) Secretion of IL-12/IL-23p40 by PsA (n = 3) and non-PsA (n = 6) SFMCs cultured for 48 h untreated (UT) or treated with DMSO control, apremilast or ADA. Data were presented as NPX values, which is an arbitrary unit on log₂ scale. Boxes and bars indicate mean and SD. * p < 0.05. ** p < 0.01. *** p < 0.001. **** p < 0.0001. ADA, adalimumab; DMSO, dimethyl sulfoxide; IL, interleukin; MCP-1, monocyte chemoattractant protein; NPX, normalized protein expression; PsA, psoriatic arthritis; SD, standard deviation; SFMC, synovial fluid mononuclear cell.

Figure 2.

(a,b) Human bone marrow mononuclear cells (n = 6) were plated and incubated with vitamin D (VitD), dexamethasone (Dex) and compounds for 7 days. TRAP-positive cells and total cells were counted. (c) SFMCs were cultured for 21 days untreated (UT), or treated with DMSO, apremilast or ADA and TRAP (n = 11) secretion was measured. (d) RAW264.7 mouse macrophages were stimulated with RANKL and compounds for 7 days and surface pitting was measured. (e) Representative photographs of osteoclast pit formation. (f) Secretion of MMP3 by FLSs were cultured for 48 h untreated (UT) or treated with DMSO control or apremilast (n = 6). (g) Human osteoblasts were cultured for 14 days untreated (UT) or treated with DMSO control or apremilast and mineralization was assessed (n = 5). Boxes and bars indicate mean and SD. * p < 0.05. ** p < 0.01. **** p < 0.0001. ADA, adalimumab; DMSO, dimethyl sulfoxide; FLS, fibroblast-like synovial cell; MMP3, matrix metalloproteinase 3; RANKL, receptor activator of nuclear factor kappa B ligand; SD, standard deviation; SFMC, synovial fluid mononuclear cell; TRAP, tartrate-resistant acid phosphatase.