SAT0013 Il-1Α and adamts5 mediated tissue damage in human cartilage explants leads to generation of tlr2 activating damps
Authors: N. Sharma, T.G. Christiansen, M. Karsdal, A.-C. Bay-Jensen, C.S. Thudium
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> The innate immune system is important for the initiation and development of joint diseases such as rheumatoid arthritis (RA) and Osteoarthritis (OA). Exogenous TLR (Toll like receptor) ligands are abundant in the synovial joints of RA and OA patients and induce the first immune response. Stimulation of TLRs results in an increased immune response and is characterised by induction of cytokines expression which in turn causes tissue damage and results in generation of more DAMPs. Hence, resulting in a self-perpetuating loop of TLR activation and TLR mediated tissue damage. <h3>Objectives</h3> The aim of this study was to investigate the effect of degradation fragments of human cartilage generated <i>ex vivo</i> or <i>in vitro</i>, on TLR-2 activation in a TLR-2 reporter cell system. <h3>Methods</h3> Human cartilage biopsies retrieved from OA patients undergoing total knee replacement at Gentofte hospital were used to generate human explants (HEX). HEX were cultured for 21 days either without (w/o) treatment or in the presence of IL-1α (10 ng/ml, 5 ng/ml and 2.5 ng/ml). Unconditioned media incubated without cartilage tissue was used as control. Tissue degradation was verified by measuring the release of aggrecan neo-epitope biomarkers AGNx1 and FFGV into the conditioned media (CM). Correspondingly, human cartilage was digested with ADAMTS5 for different time intervals (16 hour, 24 hour and 88 hour) at 37°C. The digested cartilage was removed by centrifugation and the supernatant was stored. Crushed cartilage in buffer alone was used as a control set up for each time point and buffer alone was subtracted as background. The CM and digested media (DM) was tested in the SEAP (secreted embryonic alkaline phosphatase) reporter gene based HEK hTLR2 cell line. The HEK null 1 parent cell line was used as a control. Pam3CSK4 was used as a positive control for the hTLR2 cell line. <h3>Results</h3> IL-1α induced aggrecan degradation was confirmed by increased exAGNx1 and exFFGV release in the conditioned media compared to w/o (p&lt;0.01) (figure 1a). CM from IL-1α treated explants induced a significant signal in the hTLR2 cells compared to w/o CM or to UCM with IL-1α (p&lt;0.0001). (figure 1b). Similarly, DM showed a significant induction in the hTLR2 cells after 88 hours compared to undigested human cartilage. (p&lt;0.0001) (figure 1c). <h3>Conclusions</h3> IL-1α induced cartilage degradation leads to the release of aggrecan fragments into the CM, and this CM as well as <i>in vitro</i> cleavage products from ADAMTS-5 digestion of human cartilage was able to activate the TLR2 receptor <i>in vitro</i> in a specialised reporter cell system. These data indicate that DAMPs may be released from human cartilage in the presence of pro-inflammatory cytokines and proteolytic enzymes. The released fragments can lead to TLR2 activation and cause further inflammation. These data suggest that DAMPs may play a role in the onset and maintenance of inflammation in diseases such as OA and RA. <h3>Disclosure of Interest</h3> None declared