Background Chondroitin Sulphate (CS) has shown clinical benefit in symptomatic osteoarthritis (OA). EULAR has endorsed the use of CS in OA but its mechanism is unknown. Studies have shown that high levels of CS could potentially cause precipitation of monosodium urate (MSU) crystals leading to inflammasome activation. In vitro studies have shown that CS has some anti-inflammatory effects on chondrocytes and synoviocytes from OA patients. However, the effect of CS is unclear on macrophages which are key in OA pathogenesis.
Objectives To evaluate the in vitro effects of CS on cytokine production by macrophages.
Methods THP-1 cells, a human monocytic line, were induced to differentiate into mature macrophages using a phorbol diester. To test the effects of CS on uric acid (UA) solubility, we incubated sterile solutions of CS/UA in Opti-MEM at various concentrations (CS 10-10000µg/ml and UA 10-500µg/ml). These solutions were added to the cells in vitro to monitor for pro-inflammatory effects; the solutions were also subjected to high speed centrifugal ultra-filtration to quantify, by HPLC, potential particulate formation through analysis of the remaining soluble UA in the supernatant. MSU crystals were used as a positive pro-inflammatory stimulus. Cell culture medium was removed at 24 hrs and analyzed by high-sensitivity immunoassay for IL1β. Experiments were repeated with various concentrations of a caspase-1 inhibitor (0.01µM-10 µM) to identify the component of IL1β production attributable to inflammasome activation. Finally, macrophages were pretreated with the highly purifiedbovine CS for 4 hrs prior to the addition of MSU crystals (100µg/ml) or Lipopolysaccharide (LPS) at 50µg/ml, which served as a positive control. CS doses in the physiologic range (10-200µg/ml) were used. Culture medium was collected at 24 hrs and IL1β levels were determined as above.
Results The CS/UA solutions did not lead to inflammasome activation of THP-1 macrophages (data not shown). MSU 100µg/ml consistently resulted in increased IL-1β levels by macrophages but was inhibitable by the caspase-1 inhibitor (at 1 µM or greater) confirming activation of the inflammasome as the source of this cytokine (data not shown). CS decreased IL-1 β levels in both the LPS and MSU treatment groups. Increasing doses of CS lead to a dose-dependent inhibition of IL-1β production from macrophages (Figure 1).
Conclusions High concentrations of a highly pure bovine CS do not facilitate formation of pro-inflammatory UA microparticulates or microcrystals. In contrast, CS decreases IL-1β levels in a dose-dependent manner at physiologically achievable concentrations of CS. The anti-inflammatory mechanism of CS appears compatible with inflammasome inhibition. Further research is needed to understand the means by which CS has its anti-inflammatory effect.
Acknowledgements Supported by Bioibérica (Barcelona, Spain), NIH 5T32AI007217-30 (EO) & P01 AR050245.
Disclosure of Interest E. Orlowsky Grant/research support from: Bioberica, Barcelona, Spain, T. Stabler Grant/research support from: Bioberica, Barcelona, Spain, E. Montell Employee of: Bioberica, Barcelona, Spain, J. Verges Employee of: Bioberica, Barcelona, Spain, V. Kraus Grant/research support from: Bioberica, Barcelona, Spain
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