Characterization of the interleukin-17 effect on articular cartilage in a translational model: an explorative study

Abstract

Background: Osteoarthritis (OA) is a progressive, chronic disease characterized by articular cartilage destruction. The pro-inflammatory cytokine IL-17 levels have been reported elevated in serum and synovial fluid of OA patients and correlated with increased cartilage defects and bone remodeling. The aim of this study was to characterize an IL-17-mediated articular cartilage degradation ex-vivo model and to investigate IL-17 effect on cartilage extracellular matrix protein turnover. Methods: Full-depth bovine femoral condyle articular cartilage explants were cultured in serum-free medium for three weeks in the absence, or presence of cytokines: IL-17A (100 ng/ml or 25 ng/ml), or 10 ng OSM combined with 20 ng/ml TNFα (O + T). RNA isolation and PCR analysis were performed on tissue lysates to confirm IL-17 receptor expression. GAG and ECM-turnover biomarker release into conditioned media was assessed with dimethyl methylene blue and ELISA assays, respectively. Gelatin zymography was used for matrix metalloproteinase (MMP) 2 and MMP9 activity assessment in conditioned media, and shotgun LC-MS/MS for identification and label-free quantification of proteins and protein fragments in conditioned media. Western blotting was used to validate MS results. Results: IL-17RA mRNA was expressed in bovine full-depth articular cartilage and the treatment with IL-17A did not interfere with metabolic activity of the model. IL-17A induced cartilage breakdown; conditioned media GAG levels were 3.6-fold-elevated compared to untreated. IL-17A [100 ng/ml] induced ADAMTS-mediated aggrecan degradation fragment release (14-fold increase compared to untreated) and MMP-mediated type II collagen fragment release (6-fold-change compared to untreated). MS data analysis revealed 16 differentially expressed proteins in IL-17A conditioned media compared to untreated, and CHI3L1 upregulation in conditioned media in response to IL-17 was confirmed by Western blotting. Conclusions: We showed that IL-17A has cartilage modulating potential. It induces collagen and aggrecan degradation indicating an upregulation of MMPs. This was confirmed by zymography and mass spectrometry data. We also showed that the expression of other cytokines is induced by IL-17A, which provide further insight to the pathways that are active in response to IL-17A. This exploratory study confirms that IL-17A may play a role in cartilage pathology and that the applied model may be a good tool to further investigate it.

An Evidence-Based Systematic Review of Human Knee Post-Traumatic Osteoarthritis (PTOA): Timeline of Clinical Presentation and Disease Markers, Comparison of Knee Joint PTOA Models and Early Disease Implications

“…Other reviews [ 139 , 140 ] have well described how articular cartilage explant models have used various types of injurious compression protocols to simulate mechanical injury and have demonstrated that, despite differences in protocols, these models show that they similarly and successfully induce the clinically observed pathologies following trauma to the knee joint. Hence, the mechanically-induced injury of articular cartilage leads to a significant increase in chondrocyte death, ECM loss, expression of matrix-degrading enzymes and a decrease in the COL2 gene and, to some extent, an increase in inflammatory cytokines even in the absence of inflammatory cells, such as macrophages [ 141 , 142 , 143 , 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 , 152 , 153 ]. Some studies have used a combination of mechanical injury and inflammatory cytokine treatment, typically using IL-1α, IL-1β, TNF-α, IL-17, or the combination of TNF-α/IL-6/sIL-6R [ 143 , 144 , 145 , 146 , 148 , 152 , 154 ] to mirror trauma and the presence of cytokines that are significantly present with the injured knee.…”

“…This study, like other studies on articular cartilage explants, chondrocytes and/or synoviocytes suggest that IL-17 acts synergistically with IL-1β or TNF-α [ 152 , 153 , 169 , 171 , 172 , 173 , 174 , 175 , 176 , 177 , 178 ], potentiates inflammatory gene expression (e.g., IL-6, IL-8, TNF-α) and MMP expression, and causes catabolic effects, such as ECM component loss and a decrease in the mRNA expression of chondrogenic markers, such as SOX9, XYLT1, COL2A1, and ACAN. IL-17A also caused the release of MMP-2, and -9, sGAG, the ADAMTS-mediated aggrecan degradation fragment (exAGNx1) and MMP-mediated type II collagen (C2M) in a bovine full-depth articular cartilage [ 150 ]. Moreover, this study showed that the proteomic analysis of conditioned media from IL-17A treated articular cartilage revealed an upregulation of IL-6, MMP-3, ADAMTS-4, neutrophil/macrophage chemoattractants CXCL6 and CCL20 (also known as macrophage inflammatory protein-3 alpha, MIP-3α), complement factor B, latent TGF-β-binding protein 2 and chitinase 3-like 1 (CHI3L1 or YKL-40), a secreted glycoprotein linked to OA, compared to control-treated tissue.…”

“…The * indicates the corresponding model that showed an increase of that marker in the serum (S). ( b ) Ex vivo cartilage explant models using non-0A (healthy) tissue showing the effects of blood-induced injury [ 76 , 77 , 78 ], mechanical injury using a single injurious compression without the addition of any pro-inflammatory cytokines [ 93 , 141 , 143 , 144 , 206 , 207 ], inflammatory cytokines IL-1α alone [ 144 , 149 , 155 , 156 ] or combined with injury* [ 144 , 148 ], IL-1β alone [ 147 , 152 ], TNF-α alone [ 144 , 145 , 146 , 147 ] or combined with injury* [ 143 , 144 , 145 , 146 ], IL-17A alone [ 150 ], and TNF-α/IL-6/sIL-6R alone [ 143 , 146 , 154 ] or combined with injury* [ 144 , 148 , 154 ]. (c) In vitro chondrocyte models using cells isolated from non-OA (healthy) cartilage tissue showing the effects of IL-1α [ 157 ], IL-1β [ 159 , 160 ], TNF-α [ 166 , 167 ], IL-17 or IL-17F [ 172 , 175 ], IL-6 or IL-8 [ 160 ], and LIF [ 160 , 175 ].…”

Extracorporeal Shockwave Therapy Modulates the Expressions of Proinflammatory Cytokines IL33 and IL17A, and Their Receptors ST2 and IL17RA, within the Articular Cartilage in Early Avascular Necrosis of the Femoral Head in a Rat Model

“…Recently, IL33 has been reported to be linked with IL17 in terms of contributing to immunological dysfunction in inflammatory diseases [41,42]. The IL17 receptor is a complex that consists of IL17RA, IL17RB, IL17RC, IL17RD, and IL17RE [43]. IL17RA is a key component required for IL17A activity, and blocking of IL17 binding by IL17RA could inhibit the expression of IL-6 to prevent synovial inflammation in arthritis [44].…”

“…IL17RA is a key component required for IL17A activity, and blocking of IL17 binding by IL17RA could inhibit the expression of IL-6 to prevent synovial inflammation in arthritis [44]. A high expression of IL17A 7 Mediators of Inflammation contributes to cartilage degradation by inducing disintegrinlike and metalloproteinase with thrombospondin motifs (ADAMTS) protease and matrix metalloprotease in the articular cartilage in arthritis [43]. A high expression of IL17A was observed in the hyaline cartilage, and receptor IL17RA was evaluated with regards to hypertrophy chondrocyte (Figure 6).…”