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

Abstract

Background The 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. Methods Fibroblast-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. Results PFD 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 (TGFβ), tumor necrosis factor alpha (TNFα), and interferon gamma (IFNγ) ( p = 0.022). There were no statistically significant changes in membrane expression of alpha smooth muscle actin (αSMA), intercellular adhesion molecule 1 (ICAM-1), or human leukocyte antigen DR (HLA-DR). In supernatants from FLSs stimulated with TGFβ, TNFα, and IFNγ, 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 TGFβ, TNFα, and IFNγ. Finally, PFD inhibited the deposition of hydroxyapatite by osteoblasts in a dose-dependent manner ( p = 0.0001). Conclusions PFD inhibited SpA FLS proliferation and function and osteoblast mineralization in vitro. This encourages studies of the in vivo effect of PFD in SpA.

Fibroblast-Like Synovial Cell Subsets in Rheumatoid Arthritis

“…FLS could be targeted by drugs used in fibrotic conditions such as nintedanib or pirfenidone [102]. However, these drugs are likely affecting a completely different aspect of fibroblast functions.…”

Pirfenidone attenuates synovial fibrosis and postpones the progression of osteoarthritis by anti-fibrotic and anti-inflammatory properties in vivo and in vitro

“…Previous studies have confirmed that PFD decreases autocrine expression of TGF-β and MMP-1 in human pterygium fibroblasts [ 34 ] and inhibits the production of collagen I in human intestinal fibroblasts[ 31 ]. PFD decreases the deposition of hydroxyapatite by osteoblasts in a dose-dependent manner, which may inhibit osteophyte formation [ 35 ]. Furthermore, PFD can reduce subchondral bone loss and fibrosis following murine knee cartilage injury [ 36 ].…”

Fibroblast-like synovial cell production of extra domain A fibronectin associates with inflammation in osteoarthritis

“…FLSs were then seeded at a concentration of 5.0 × 10 4 cells/mL in DMEM and antibiotics supplemented with 5% FBS and incubated for 24 h at 37 °C. The cells were either untreated or stimulated with TGFβ1 at 5 ng/ml, TNFα (PeproTech) at 10 ng/mL, lipopolysaccharide (LPS) (Sigma) at 100 ng/ml, or IL-6 (PeproTech) at 10 ng/ml as done previously [25–27]. Cells were fixed with 4% paraformaldehyde and then incubated for 30 min at room temperature with PBS with 0.05% Tween20, 1% bovine serum albumin, 5% normal goat serum, and 0.03% NaN 3 .…”