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.
Objectives
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.
Methods
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 35SO4. Alternatively, explants were pre-labelled for 4 hour with 35SO4 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.
Results
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 35SO4 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 35SO4-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.
Conclusions
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.
In healthy cartilage, there is a balance between anabolic and catabolic activities of chondrocytes that maintains the functional integrity of the extracellular matrix. However, during osteoarthritis (OA), chondrocytes become more catabolically active and express increased levels of matrix degrading enzymes, such as MMPs and ADAMTSs. Increased MMP and ADAMTS activity results in a net loss of the extracellular matrix and therefore leads to cartilage damage. Previously, we found that the anti-inflammatory cytokine Interleukin 37 (IL37) is able to counter-regulate the catabolic status of chondrocytes by reducing the IL1β-driven expression of pro-inflammatory cytokines and catabolic enzymes.
Objectives
The goal of this study was to investigate, in human OA cartilage explants, the effect of IL37 on sulfated glycosaminoglycans (GAG) content and synthesis of extracellular matrix molecules and cartilage degrading enzymes to investigate its therapeutic potential in OA.
Methods
Human cartilage was obtained from eighteen OA patients undergoing total knee or hip arthroplasty. Biopsy punches of 4 mm in diameter were made to equalize explant size. After culturing overnight, explants were incubated for 48 h with three doses (1, 10 or 100 ng/ml) of recombinant-human IL37 (rhIL37). In the supernatant of the explant cultures, GAG release was measured with the DMB assay and levels of the ARGS neoepitope, which is one of the products of aggrecan degradation by ADAMTS5, were detected using Western Blot. Furthermore, gene and protein expression of extracellular matrix molecules and cartilage degrading enzymes were measured. Nitric oxide (NO), an important effector molecule that may suppress cartilage matrix synthesis, levels were measured in the supernatant of the explants culture using Griess reagens.
Results
Adding rhIL37 (100 ng/ml) to OA cartilage explants caused a highly significant reduction in GAG release to the supernatant of, on average, 32% in eighteen donors (Figure 1). Gene expression of the matrix molecules aggrecan and collagen type II was not affected, indicating that this effect of rhIL37 was not due to a loss of aggrecan synthesis. Another mechanism to prevent GAG release in cartilage is via inhibition of NO synthesis, but NO levels in the supernatant were comparable between rhIL37 treated groups and the control group. In contrast, after addition of rhIL37, ARGS neoeptiope levels, which reflect ADAMTS5 activity, were dose-dependently down regulated in the supernatant. Furthermore, protein analysis of the explants showed that rhIL37 reduced ADAMTS5 levels. These data indicate that IL37 interferes with the amount active matrix degrading enzymes in the cartilage matrix. However, gene expression of ADAMTS5 was not affected by rhIL37, indicating that the effect of IL37 on ADAMTS5 is post translational.
Conclusions
Our data show that rhIL37 reduces GAG release by OA cartilage explants. The mechanism behind this protective effect of IL37 probably runs via a reduction in ADAMTS5 abundance in the cartilage matrix, which is the main aggrecanase involved in OA This effect of IL37 on ADAMTS5 is probably post translational. Our data indicate that IL37 can maintain cartilage matrix integrity under OA conditions and is able to reduce the severity of cartilage destruction during OA.
TGF-β1 is an important growth factor to promote the differentiation of T helper 17 (Th17) and regulatory T cells (Treg). The potential of TGF-β1 as therapeutic target in T cell-mediated diseases like rheumatoid arthritis (RA) is unclear. We investigated the effect of TGF-β1 inhibition on murine Th17 differentiation in vitro, on human RA synovial explants ex vivo, and on the development of experimental arthritis in vivo. Murine splenocytes were differentiated into Th17 cells, and the effect of the TGF-βRI inhibitor SB-505124 was studied. Synovial biopsies were cultured in the presence or absence of SB-505124. Experimental arthritis was induced in C57Bl6 mice and treated daily with SB-505124. Flow cytometry analysis was performed to measure different T cell subsets. Histological sections were analysed to determine joint inflammation and destruction. SB-505124 potently reduced murine Th17 differentiation by decreasing Il17a and Rorc gene expression and IL-17 protein production. SB-505124 significantly suppressed IL-6 production by synovial explants. In vivo, SB-505124 reduced Th17 numbers, while increased numbers of Tregs were observed. Despite this skewed Th17/Treg balance, SB-505124 treatment did not result in suppression of joint inflammation and destruction. Blocking TGF-β1 signalling suppresses Th17 differentiation and improves the Th17/Treg balance. However, local SB-505124 treatment does not suppress experimental arthritis.
