Background 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).

Objectives 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.

Methods Culture: The THP-1 human monocytic cell line (ATCC TIB-202) was grown in the recommended media to 1.5 x10⁶ cells/ml, plated on a 96 well plate at 2 x 10⁴ 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 x10⁶ 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, H₂O 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 25cm² flasks with activity measured on fresh lysate derived from 2x10⁶ cells.

Assays: Media and cell contents were assayed for IL-1β and proIL-1β by ELISA (R&D Systems). Caspase-1 activity was measured by fluorometric assay (R&D Systems). Cell viability was measured using PrestoBlue reagent (Invitrogen).

Analysis: 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 Dunnett's post-hoc test and post-hoc linear trend were done using Graphpad Prism software.

Results As expected, HA fragments produced large increases (p<0.0001) in IL-1β release with shorter fragments (10 and 100 μg/ml) inducing more IL-1 β than larger fragments (p<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<0.0001) in intracellular caspase-1 activity, CS had no effect on this activity. CS significantly reduced (p<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).

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Conclusions 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.

References

1. Stern R, Eur J Cell Biol 2004,83:317.

2. Orlowsky E, BMC Musculoskeletal Disord 2014,15:318

Disclosure of Interest T. Stabler Grant/research support from: Bioiberica, E. Montell Employee of: Bioiberica, J. Verges Employee of: Bioiberica, V. Kraus Grant/research support from: Bioiberica