SAT0029 Attenuation of Hyaluronan Fragment Induced Inflammatory Response in Macrophages by Chondroitin Sulphate
Authors: T. Stabler, E. Montell, J. Verges, V.B. Kraus
Journal: Annals of the Rheumatic Diseases (2015)

<h3>Background</h3> Hyaluronan (HA) fragments are known to be pro-inflammatory and able to induce an inflammatory response from macrophages characterized by the release of IL-1β and other pro-inflammatory cytokines (1). We have previously shown that pharmaceutical grade chondroitin sulphate (CS) can attenuate the monosodium urate crystal mediated release of inflammatory cytokines from activated macrophages (2). <h3>Objectives</h3> Determine if CS could similarly ameliorate an HA fragment mediated inflammatory response and if any CS inflammatory inhibition was acting at the level of the inflammasome. <h3>Methods</h3> <i>Culture:</i> The THP-1 human monocytic cell line (ATCC TIB-202) was grown in the recommended media to 1.5 x10<sup>6</sup> cells/ml, plated on a 96 well plate at 2 x 10<sup>4</sup> cells/well, then induced to differentiate into mature macrophages by the addition of 200nM of phorbol 12-myristate 13-acetate (PMA). After 2 days the media was replaced without the PMA and grown for 2 more days. Cells were primed with 10 ng/ml of LPS for 24 hrs with CS in various physiologically relevant concentrations (0, 10-200 μg/ml). After 24 hrs, cells were washed with PBS and media replaced with serum free Opti-MEM along with the previously mentioned concentrations of CS and various molecular weights (MW) (sizes) (ultra-low MW=7.5 kDA, low MW=29 kDa, medium MW=289 kDA, and high MW=1.54 x10<sup>6</sup> kDa) and concentrations (0, 1, 10, and 100 ug/ml) of HA fragments. After a further 24 hrs, cell viability was assessed and the media harvested. Following a PBS wash, H<sub>2</sub>O was added and plates were subjected to 3 sonication/freeze/thaw cycles in order to release the cell contents. Media and cell contents were snap frozen until further analysis. Caspase-1 experiments were done in 25cm<sup>2</sup> flasks with activity measured on fresh lysate derived from 2x10<sup>6</sup> cells. <i>Assays:</i> Media and cell contents were assayed for IL-1β and proIL-1β by ELISA (R&amp;D Systems). Caspase-1 activity was measured by fluorometric assay (R&amp;D Systems). Cell viability was measured using PrestoBlue reagent (Invitrogen). <i>Analysis:</i> After normalization for cell viability, all results were expressed as fold change from the negative control (media only without CS or HA). One-way ANOVA with Dunnett9s post-hoc test and post-hoc linear trend were done using Graphpad Prism software. <h3>Results</h3> As expected, HA fragments produced large increases (p&lt;0.0001) in IL-1β release with shorter fragments (10 and 100 μg/ml) inducing more IL-1 β than larger fragments (p&lt;0.0001 for gradient effect); no proinflammatory effect was observed for high MW HA. CS (100-200 μg/ml) produced a dose dependent reduction in IL-1β release in cells treated with 10 μg/ml of the 3 HA lower MW fragments. While ultra-low MW HA fragments induced a significant increase (p&lt;0.0001) in intracellular caspase-1 activity, CS had no effect on this activity. CS significantly reduced (p&lt;0.05) intracellular IL-1β and proIL-1β in cells treated with ultra-low MW HA but the ratio between the 2 was unchanged (figure 1). <h3>Conclusions</h3> HA fragments (≤289 kDa) induced an inflammatory response in THP-1 macrophages that could be attenuated by physiologically achievable concentrations of CS. Since we did not observe a decrease in intracellular caspase-1 activity due to CS, it can be concluded that the anti-inflammatory effect of CS is upstream of the inflammasome. <h3>References</h3> Stern R, Eur J Cell Biol 2004,83:317. Orlowsky E, BMC Musculoskeletal Disord 2014,15:318 <h3>Disclosure of Interest</h3> T. Stabler Grant/research support from: Bioiberica, E. Montell Employee of: Bioiberica, J. Verges Employee of: Bioiberica, V. Kraus Grant/research support from: Bioiberica