Free Fatty Acids in Bone Pathophysiology of Rheumatic Diseases

Abstract

Obesity-in which free fatty acid (FFA) levels are chronically elevated-is a known risk factor for different rheumatic diseases, and obese patients are more likely to develop osteoarthritis (OA) also in non-weight-bearing joints. These findings suggest that FFA may also play a role in inflammation-related joint damage and bone loss in rheumatoid arthritis (RA) and OA. Therefore, the objective of this study was to analyze if and how FFA influence cells of bone metabolism in rheumatic diseases. When stimulated with FFA, osteoblasts from RA and OA patients secreted higher amounts of the proinflammatory cytokine interleukin (IL)-6 and the chemokines IL-8, growth-related oncogene α, and monocyte chemotactic protein 1. Receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin, and osteoblast differentiation markers were not influenced by FFA. Mineralization activity of osteoblasts correlated inversely with the level of FFA-induced IL-6 secretion. Expression of the Wnt signaling molecules, axin-2 and β-catenin, was not changed by palmitic acid (PA) or linoleic acid (LA), suggesting no involvement of the Wnt signaling pathway in FFA signaling for osteoblasts. On the other hand, Toll-like receptor 4 blockade significantly reduced PA-induced IL-8 secretion by osteoblasts, while blocking Toll-like receptor 2 had no effect. In osteoclasts, IL-8 secretion was enhanced by PA and LA particularly at the earliest time point of differentiation. Differences were observed between the responses of RA and OA osteoclasts. FFA might therefore represent a new molecular factor by which adipose tissue contributes to subchondral bone damage in RA and OA. In this context, their mechanisms of action appear to be dependent on inflammation and innate immune system rather than Wnt-RANKL pathways.

Keywords: fatty acid; inflammation; osteoarthritis; osteoblasts; osteoclasts; rheumatoid arthritis.

Figure 1

Free fatty acids palmitic acid and linoleic acid induce a proinflammatory response in osteoblasts. Supernatants of RA and OA osteoblasts were analyzed after 24 h stimulation with palmitic acid or linoleic acid for IL-6 (RA: n = 14, OA: n = 20), IL-8 (RA: n = 14, OA: n = 20), and MCP-1 (RA: n = 3, OA: n = 3). Both fatty acids significantly enhanced the secretion of these factors in RA (A–C) and OA (D–F) osteoblasts. *p < 0.05; **p < 0.001; and ***p < 0.001. MCP-1, monocyte chemotactic protein 1 (CCL2).

Figure 2

Free fatty acids palmitic acid and linoleic acid induce the secretion of the chemokine GRO-α in osteoblasts. GRO-α was induced from a non-detectable level in RA (A,B, n = 14) and OA (C,D, n = 20) in a subset of osteoblast populations. *p < 0.05; **p < 0.001; and ***p < 0.001. GRO-α, Growth-related oncogene α (CXCL1).

Figure 3

Blocking Toll-like receptor (TLR) 4 but not TLR2 significantly reduces the palmitic-acid-mediated interleukin-8 (IL-8) secretion by osteoblasts. TLR2 and TLR4, two receptors for free fatty acid (FFA), were blocked by neutralizing antibodies on rheumatoid arthritis (RA) osteoblasts (n = 3). Palmitic-acid-induced IL-8 secretion was significantly reduced by the anti-TLR4 antibody but not by the anti-TLR2 antibody or the isotype control antibody. *p < 0.05. Ctrl, control; Ab, antibody; TLR, Toll-like receptor.

Figure 4

Mineralization activity of osteoblasts correlates with their IL-6 secretion. The mineralization activity of rheumatoid arthritis (RA) (n = 10) and osteoarthritis (OA) (n = 14) osteoblasts was inversely correlated with the changes in IL-6 secretion levels. There was a significant inverse correlation for RA (A) and OA osteoblasts (B) as well as the combination of both (C).

Figure 5

Human osteoclasts and their precursors from rheumatoid arthritis (RA) patients respond differently to free fatty acids than their counterparts from osteoarthritis (OA) patients. Palmitic acid and linoleic acid significantly increased the interleukin-8 (IL-8) secretion of RA osteoclasts and their precursors (n = 4), particularly at the earliest time point (d8) (A–C), while equivalent cells from OA patients (n = 4) only showed weaker effects without statistical significance (D–F). *p < 0.05; **p < 0.001; and ***p < 0.001.