Abstract
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 (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).ConclusionsPFD inhibited SpA FLS proliferation and function and osteoblast mineralization in vitro. This encourages studies of the in vivo effect of PFD in SpA.
Highlights
The pathogenesis of spondyloarthritis (SpA) involves both inflammation and new bone formation in the spine
One of the distinct features discriminating spondyloarthritis from rheumatoid arthritis is transforming growth factor beta (TGFβ) induced upregulation of myofibroblasts involved in new bone formation at entheseal sites [4, 14]
Study subjects A study population consisting of SpA patients (n = 6) with peripheral involvement was included for obtaining synovial fluid for growing fibroblast-like synoviocytes (FLSs)
Summary
FLSs were stimulated with either TGFβ at 5 ng/ml, TNFα at 10 ng/ml, and IFNγ at 10 ng/ml alone or in combination and cultured with or without PFD at 1.0 mg/ml for 48 h This concentration has previously been shown not to induce apoptosis or cell death in fibroblasts from several anatomical sights including FLSs [22,23,24,25]. Flow cytometry SpA FLSs were cultured at a density of 5.0 × 104 cells/ml in RPMI-1640 (Lonza) supplemented with 10% FBS, penicillin, streptomycin, and glutamine at 37 °C and 5% CO2 in a humidified incubator for 24 h. Cells were cultured in osteoblast mineralization medium (C-27020, PromoCell) with PFD either at 0.25 mg/ml, 0.5 mg/ml, or 1.0 mg/ml for 14 days. Calculations and graphs were made with Stata (StataCorp LP) and GraphPad Prism (GraphPad Software)
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