AB0092 Il37 inhibits proteoglycan loss in human oa cartilage: link between il37 and mmp3
Authors: E. Van Geffen, A. van Caam, C. Thudium, A.-C. Bay-Jensen, E. Blaney Davidson, P. van der Kraan
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> Glycosaminoglycans (GAGs) are essential for the pressure-resistant function of cartilage. During osteoarthritis (OA), GAGs are lost from cartilage. This loss impairs the functional and structural integrity of cartilage, thereby accelerating further cartilage damage. Proteoglycan degradation is mediated by enzymes such as MMP3, MMP13 and ADAMTS5. <h3>Objectives</h3> Recently we discovered that interleukin 37 (IL37) lowers the expression of these enzymes in human OA chondrocytes. The goal of this study was to investigate if IL37 protects against GAG loss in freshly obtained human OA explants. <h3>Methods</h3> Human cartilage was obtained from ten OA patients undergoing total knee or hip arthroplasty. Per condition 6 cartilage explants of 4 mm in diameter were used. Explants were incubated with recombinant IL37 (rhIL37) for up to 6 days. Every other day new rhIL37 was added. Additionally, an MMP3-inhibitor, or MMP13-inhibitor or ADAMTS5-inhibitor was added in the same protocol. Sulfated GAGs (sGAGs) were visualised by histology, and sGAG release in culture medium was quantified using 1,9-dimethylmethylene blue. To study sGAG synthesis, explants were incubated with rhIL37 followed by 4 hour labelling with <sup>35</sup>SO<sub>4</sub>. Alternatively, explants were pre-labelled for 4 hour with <sup>35</sup>SO<sub>4</sub> followed by incubation with rhIL37 to study sGAG degradation. Additionally, expression levels of proteoglycans and cartilage matrix degrading enzymes were measured by qPCR and Western Blot. Activity of MMP and ADAMTS enzymes was determined by measuring FFGV, ARGS and NITEGE neo-epitope levels in the supernatant of the cultures, using ELISA. <h3>Results</h3> Culturing human OA explants for up to 6 days, caused a reduction in GAG content as observed by loss of Safranin O staining. In addition, a release of sGAGs was measured in the supernatant of on average 17,5 µg/ml per mg cartilage. Incubation of cartilage explants with rhIL37 significantly reduced the release of GAGs. A maximal reduction of 24% was already observed with the lowest dose of rhIL37 (1 ng/ml). No effect of rhIL37 on the amount of incorporated <sup>35</sup>SO<sub>4</sub> was observed, indicating that rhIL37 does not alter the synthesis of GAGs. This is supported by the observation that rhIL37 does not affect the mRNA expression of the large proteoglycans and SLRPS. In contrast, we did find that rhIL37 significantly reduced the loss of <sup>35</sup>SO<sub>4</sub>-labelled GAGs from cartilage explants. In addition, rhIL37 reduced MMP3 and MMP13 protein expression and lowered both MMP and ADAMTS mediated degradation of proteoglycan fragments: FFGV neo-epitope and ARGS and NITEGE neo-epitopes, confirming that rhIL37 reduces active degradation of sGAGs. Lastly, to investigate which proteolytic enzyme contributes to the sGAG release in our culture system, a MMP3-, MMP13- or ADAMTS5-inhibitor was added to the explants. Strikingly, we found that only MMP3 inhibition mimicked IL37 function, suggesting that the effects of rhIL37 run via MMP3. <h3>Conclusions</h3> Our data show that rhIL37 reduces sGAG release of cartilage explants, indicating that IL37 supports cartilage matrix integrity. To our knowledge this is the first report demonstrating this anti-destructive effect of IL37 on freshly obtained human OA cartilage explants. Possible these effects run via MMP3 because IL37 reduced MMP3 expression and only MMP3 inhibition results in similar effects as rhIL37 addition. <h3>Disclosure of Interest</h3> None declared