FRI0523 TLR-1/2 SIGNALING IMPAIRS MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION IN CHONDROCYTES VIA THE INDUCTION OF NITRIC OXIDE
Authors: Ping Shen, Michael Fuchs, Marie Reisener, Clemens Gwinner, Peihua Wu, Tobias Jung, Matthias Pumberger, Carsten Perka, Max Löhning
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background</h3> Osteoarthritis (OA) is a degenerative disease that causes progressive loss of joint function, representing a severe health problem plaguing this ageing world. Cartilage matrix degradation and catabolic factor production are closely associated with OA clinical symptoms<sup>1</sup>. Recent studies suggest that, metabolism is important for maintaining cartilage function, and aberrant metabolic activities in chondrocytes have been shown in various OA animal models and patients<sup>2</sup>. Despite the severity of OA, the initial triggers of this disease are mostly unknown. Small molecules generated during physiological catabolic reactions often function as damage-associated molecular patterns (DAMP) to activate innate immunity through receptors such as Toll-like receptors (TLR), resulting in enhanced expression of matrix metalloproteinases (MMP) and catabolic factors<sup>3</sup>. Here we studied the effect of various TLR signaling on chondrocyte protein production and mitochondrial oxidative phosphorylation (OXPHOS). We furthermore establish connections between catabolic factors and altered OXPHOS of chondrocytes, aiming to understand the underlying molecular mechanisms. <h3>Objectives</h3> This project is designed to investigate the roles of TLR signaling in the development of OA and the involved molecular mechanisms <h3>Methods</h3> Chondrocytes were obtained from the femural condyles of OA patients that underwent total knee arthroplasty in the Charité hospital. 3D spheroids were generated and cultured under physioxia (4.2% O2) with addition of TGF-β3. PGN, PolyI:C, LPS, Flagellin, FSL-I, Imiquimod, ssRNA, or CpG were used as agonist of TLR-1/2, -3, -4, -5, -2/6, -7, -8, or -9, respectively. Supernatants were collected, and culture medium were renewed twice per week. Pellets were weighted and processed for Alcian blue staining, or mRNA detection, or OXPHOS assay using Seahorse XFe96 Spheroid Mito Stress Test Kit. We used Griess reaction to quantify nitric oxide (NO) in the supernatant and L-NAME to block NO production. We used One-way ANOVA and t test for statistical analysis. <h3>Results</h3> TLR-1/2 and TLR-2/6 stimulation drastically impaired the cartilage matrix production, as they imposed the slowest pellet growth, lowest expression of matrix proteins COL2 and AGC1, but highest expression of matrix degrading enzymes MMP3, ADAMTS5, and the catabolic factor NO. Remarkably, this phenomenon is associated with drastically diminished OXPHOS activity, as shown by the reduction of both basal and maximal respiratory capacity. Moreover, blockade of NO production reversed the adverse effect imposed by TLR-1/2 stimulation, namely restored their OXPHOS activities and Col2 and AGC1 production in a dose-dependent manner. <h3>Conclusion</h3> Among the whole spectrum of TLR signaling, stimulation of TLR-1/2 and -2/6 impose the strongest inhibition on chondrocyte pellet growth by suppressing matrix protein synthesis and facilitating matrix degradation. TLR-1/2 stimulation causes the reduction of OXPHOS capacity, which is mediated by the induction of NO. Thus, TLR-1/2 stimulation, via inducing NO production, impairs the mitochondrial respiration in chondrocytes and thus possibly promotes the development of OA. <h3>References</h3> [1] Piotr Wojdasiewicz. et al. The Role of Inflammatory and Anti-Inflammatory Cytokines in the Pathogenesis of Osteoarthritis. Mediators Inflamm. 2014: 561459. [2] Mobasheri A. et al. The role of metabolism in the pathogenesis of osteoarthritis. Nat Rev Rheumatol. 201713(5):302 [3] Orlowsky EW, Kraus VB. The role of innate immunity in osteoarthritis: when our first line of defense goes on the offensive. J Rheumatol. 201542(3):363 <h3>Disclosure of Interests</h3> Ping Shen: None declared, Michael Fuchs: None declared, Marie Reisener: None declared, Clemens Gwinner: None declared, Peihua Wu: None declared, Tobias Jung: None declared, Matthias Pumberger: None declared, Carsten Perka Consultant for: DePuy/Synthes; Link; Smith&amp;Nephew; Zimmer, Max Löhning: None declared