The antifibrotic drug pirfenidone inhibits spondyloarthritis fibroblast-like synoviocytes and osteoblasts in vitro

BackgroundThe pathogenesis of spondyloarthritis (SpA) involves both inflammation and new bone formation in the spine. In line with this, the disease has been characterized as both inflammatory and fibrotic. The current treatment dampens inflammation while new bone formation can progress. Therefore, there is an unmet therapeutic need for the treatment of new bone formation in SpA. Fibrosis is mediated by myofibroblasts and new bone formation is the result of increased osteoblast mineralization and decreased osteoclast resorption. Here, we evaluate the potential effect of the newly approved anti-fibrotic agent pirfenidone (PFD) on fibrosis and new bone formation in cell culture models of SpA.MethodsFibroblast-like synoviocytes (FLSs) were isolated from SpA patients (n=6) while the osteoblast cell line Saos-2 was purchased. The cells were cultured with PFD at 0.25 0.5, or 1.0 mg/ml. The proliferation of FLSs was analyzed with light microscopy and flow cytometry. The differentiation and activation of FLSs was assessed with flow cytometry, a membrane-based antibody array and enzyme-linked immunosorbant assays. The mineralization capacity of osteoblasts was studied with an assay measuring deposition of hydroxyapatite.ResultsPFD reduced the Ki67 expression 7.1-fold in untreated FLSs (p=0.001) and 11.0-fold in FLSs stimulated with transforming growth factor beta (TGF beta), tumor necrosis factor alpha (TNF alpha), and interferon gamma (IFN gamma) (p=0.022). There were no statistically significant changes in membrane expression of alpha smooth muscle actin (alpha SMA), intercellular adhesion molecule 1 (ICAM-1), or human leukocyte antigen DR (HLA-DR). In supernatants from FLSs stimulated with TGF beta, TNF alpha, and IFN gamma, PFD decreased the secretion of 3 of 12 proteins more than 2-fold in the membrane-based antibody array. The changes in secretion of monocyte chemoattractant protein 1 (MCP-1) and chitinase-3-like protein 1 (CHI3L1, YKL-40) were validated with ELISA. PFD decreased the secretion of both Dickkopf-related protein 1 (DKK1) (p=0.006) and osteoprotegerin (OPG) (p=0.02) by SpA FLSs stimulated with TGF beta, TNF alpha, and IFN gamma. Finally, PFD inhibited the deposition of hydroxyapatite by osteoblasts in a dose-dependent manner (p=0.0001).ConclusionsPFD inhibited SpA FLS proliferation and function and osteoblast mineralization in vitro. This encourages studies of the in vivo effect of PFD in SpA.