FRI0037 Chondroitin Sulphate Inhibition of NF-κB Activity: Attentuation of Hyaluronan Fragment Induced Inflammatory Response in Macrophages by Chondroitin Sulphate Is Mediated by Reduction in NF-κB Activity
Authors: T. Stabler, E. Montell, J. Vergés, V.B. Kraus
Journal: Annals of the Rheumatic Diseases (2016)

<h3>Background</h3> NF-κB activation serves as a master regulatory switch for an inflammatory response in macrophages. Chondroitin sulphate (CS), a natural glycosaminoglycan of the cartilage extracellular matrix, has been shown to have clinical benefit in osteoarthritis (OA) producing anti-inflammatory and positive effects on the joint (1). We have previously shown that hyaluronan (HA) fragments induce an inflammatory response, in the form of IL-1β release, in macrophages that have been primed with the Toll-Like Receptor 4 (TLR4) agonist, lipopolysaccharide (LPS) and that CS can attenuate this response. We further demonstrated that this attenuation by CS occurred upstream of the inflammasome (2). <h3>Objectives</h3> Determine if this inhibition is acting at the level of NF-κB activation. <h3>Methods</h3> The THP1-Lucia NF-κB reporter monocyte cell line (InvivoGen) was grown as recommended. 10<sup>5</sup> cells/well were plated along with varying concentrations of CS (0, 10–200 μg/ml) and either 1 μg/ml LPS or 10<sup>7</sup> heat killed listeria monocytogenes (HKLM). In a 2nd experiment, cells were set up similarly but with the addition of 10 μg/ml ultralow molecular weight (ULMW) HA. In a 3rd experiment cells were set up as above with ULMW HA but substituting a caspase-1 inhibitor (EMD Millipore,10 μM) for CS. After 24 hours, media were tested for the NF-κB activity using QUANTI-Luc reagent (Invivogen) and for cell viability using PrestoBlue reagent (Invitrogen). After normalizing for cell viability, all results were expressed as fold change from the media only control. One-way ANOVA with Dunnett9s post-hoc test and inhibition dose response curves were performed. <h3>Results</h3> NF-κB activity increased significantly (p&lt;0.0001) in THP1-Lucia NF-κB reporter monocytes treated with either TLR4 or TLR2 agonists (i.e. 1 μg/ml of LPS or 10<sup>7</sup> HKLM bacteria). CS (50–200 μg/ml) produced a dose dependent (p=0.02 to p=0.0005) reduction in NF-κB activity in LPS treated cells. In HKLM treated cells, CS (100–200 μg/ml) also produced a similar dose dependent (p=0.004 to p=0.01) reduction in NF-κB activity. Dose response curves indicated a 50% inhibition concentration (IC50) of 146 μg/ml CS for LPS (●) stimulated cells and 64 μg/ml for HKLM (▲) stimulated cells (Fig. 1A). HA fragments alone produced no increase in NF-κB activity but were found to have an additive effect through either the TLR4 or TLR2 pathway (i.e. LPS or HKLM). The combination of TLR activation and HA produced greater NF-κB activity than either LPS or HKLM alone (p&lt;0.0001**** and p=0.0017***, Fig. 1A). CS (200 μg/ml) was still an effective inhibitor under these conditions (p=0.045* and p=0.02**, Fig. 1B). Inhibiting caspase-1 activity, and thus blocking IL-1β production, completely blocked the additive effect of HA fragments (↓, Fig. 1C). Caspase inhibition had no effect on LPS or HKLM alone. <h3>Conclusions</h3> The inflammatory response induced by HA fragments in the presence of TLR2 or TLR4 activation can be attenuated by CS at physiologically achievable concentrations via inhibition of NF-κB activation. HA9s synergistic effect on NF-κB is due to positive feedback through inflammasome mediated active IL-1β production. These data provide a plausible explanation for the clinical effects of CS in OA. <h3>References</h3> du Souich P, Pharmacol Ther 2014, 142:362. Stabler T, Osteoarthritis Cartilage 2015, 23:A263. <h3>Disclosure of Interest</h3> T. Stabler Grant/research support from: Bioiberica, E. Montell Employee of: Bioiberica, J. Vergés Employee of: Bioiberica, V. Kraus Grant/research support from: Bioiberica