Effect of Tumor Necrosis Factor Inhibitor Therapy on Osteoclasts Precursors in Rheumatoid Arthritis

Abstract

Objective. Tumor necrosis factor (TNF) increases circulating osteoclast (OC) precursors numbers by promoting their proliferation and differentiation. The aim of this study was to assess the effect of TNF inhibitors (TNFi) on the differentiation and activity of OC in rheumatoid arthritis (RA) patients. Methods. Seventeen RA patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers, in vitro OC differentiation assays, and qRT-PCR for OC specific genes was performed. Results. After TNFi therapy, patients had reduced RANKL surface expression in B-lymphocytes and the frequency of circulating classical CD14^brightCD16^- monocytes was decreased. Serum levels of sRANKL, sRANKL/OPG ratio, and CTX-I were reduced in RA patients after TNFi treatment. Moreover, after exposure to TNFi, osteoclast differentiation and activity were decreased, as well as the expression of TRAF6 and cathepsin K. Conclusion. We propose that TNFi arrests bone loss and erosion, through two pathways: direct reduction of osteoclast precursor numbers and inhibition of intracellular signaling pathways acting through TRAF6.

Figure 1

Functional assays of in vitro differentiated OC. (a) Representative images, at culture day 21, of adhering precursors stimulated with M-CSF, RANKL, dexamethasone, and TGF-β stained for TRAP, where the pit assay was performed. (b) OC number increased throughout time and, at culture days 14 and 21, patients at follow-up had significantly fewer osteoclasts than at baseline (p = 0.0094 and 0.0203, resp.). No differences were found in the number of resorption pits/mm²; patients at follow-up had significantly smaller pits at culture day 21 (resorbed area/pit, p = 0.0038) and significantly less resorbed area at culture day 21, when compared to their baseline (p = 0.0383). Dots represent median counts for each group at each time point and bars represent interquartile range. d: day; OC: osteoclast. Scale bars: 100 μm; red arrows: osteoclasts; black arrows: resorption pits. τ: remained significant after adjusting for multiple comparisons.

Figure 2

Gene expression profile of stimulated adhering precursors in culture for 21 days. At day 1, TRAF6 expression in patients at follow-up was significantly reduced (p = 0.0229). At day 7, both FRA-2 and CTSK expressions were significantly decreased (p = 0.0242 and 0.035, resp.). At day 21, patients at follow-up had significantly reduced expression when compared to patients at baseline (p = 0.008). Gene expression shown as a ratio to housekeeping expression (2(−ΔCT)/2(−ΔCT)). Dots in graphs represent median gene expression for each group at each time point and lines represent interquartile range [25–75]. d: day; TRAF6: gene encoding tumor necrosis factor receptor-associated factor-6; FRA-2: gene encoding Fos-related antigen-2; CTSK: gene encoding cathepsin K. τ: remained significant after adjusting for multiple comparisons.