Auteurs:
Elodie Zocola | HUG - Geneva University Hospitals/ UNIGE | Switzerland
Dr. Félix Renaudin | Faculty of Medicine, UNIGE
Cynthia Diabanza | Faculty of Medicine, UNIGE
Dr. Thomas Laumonier | Faculty of Medicine, UNIGE
Didier Hannouche | HUG - Geneva University Hospitals/ UNIGE | Switzerland
Introduction:
In osteoarthritis (OA), articular cartilage follows progressive degeneration, where low-grade inflammation plays a pivotal role via exposure to reactive oxygen species (ROS). In chondrocytes, the only cells in cartilage, NADPH oxidase 4 (NOX4) is a major producer of ROS, significantly impacting regulation of catabolism. Our prior study showed that NOX4 deletion reduces cartilage degradation and regulates the anabolism/catabolism balance. As IL-1β is a key cytokine involved in OA, we hypothesize that NOX4 may influence the IL-1β secretion pathway through the NLRP3 inflammasome. This study evaluates the mechanisms of NOX4-induced OA in mice.
Methods:
OA was induced in vivo by destabilization of the medial meniscus (DMM) in WT and NOX4-/- mice. After 8 weeks, mice were sacrificed for immuno-histo-chimistry (IHC) analysis. Femoral heads from 9/10-week-old mice were cultured for 72 hours with PBS or IL-1β to induce OA ex vivo. Primary chondrocytes (PC) extracted from 4/6-day-old pups femoral heads and knees were cultured for 24 hours with PBS or IL-1β to induce OA in vitro. IL-6, IL-18, and MMP3 were quantified in ex vivo and in vitro supernatants by ELISA. Sulfated glycosaminoglycans (sGAG) concentration was assessed in supernatants by colorimetric dosage. Genes expressions of PC were quantified by RTqPCR.
Results:
IHC revealed stronger IL-1β and NLRP3 expression after DMM surgery in WT compared to NOX4-/- mice. In PC supernatant, we observed an IL-18 increase post IL-1β in WT but not in NOX4-/-.
In WT, RTqPCR showed a decreased anabolism genes transcription (AGG, COL2, SOX9) and NOX4 and increased catabolism and inflammation genes transcription (MMP3, MMP13, ADAMTS4, ADAMTS5, and IL-1, IL-6, TNF, COX2) post IL-1β.
In ex vivo supernatants, we observed increased IL-6 and sGAG concentration post IL-1β. In vitro, we observed increased MMP3, IL6, and decreased sGAG concentration in supernatants post IL-1β.
In NOX4-/- compared to WT, post IL1-β, we observed lower ADAMTS4/5 and IL1-β transcription, higher COX2 and IL6 transcription, but no IL6 concentration increase in supernatant. In the PC supernatants, we found a lower sGAG concentration.
Conclusion:
We demonstrate that NOX4 deletion inhibits NLRP3 inflammasome activation and IL-18 secretion. We showed that NOX4 deficiency inhibits ADAMTS gene expression. These results underline that NOX4 could aggravate OA by activating ADAMTS enzymes via the NLRP3/IL-18 pathway.