SAT0067 Role of nox2-derived reactive oxygen species in s100a8/a9-driven inflammatory osteoarthritis
Authors: N. Kruisbergen, S. van Dalen, M. van den Bosch, A. Blom, P. van Lent
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> Synovitis in inflammatory osteoarthritis (OA) is driven by locally released DAMPs like S100A8/A9 proteins that have been shown to enhance joint destruction. S100A8/A9 induce ROS (reactive oxygen species) release by phagocytes in OA synovium via the NADPH-oxidase 2 (NOX2)-complex. Assembly of this complex is dependent on the neutrophils cytosolic factor (Ncf1). In this complex, the NOX2 protein is responsible for ROS production. <h3>Objectives</h3> In the present study we investigated whether NOX2-derived ROS are involved in joint pathology during collagenase-induced OA (CiOA). <h3>Methods</h3> CiOA was induced in knee joints of wild type (WT) and Ncf1-deficient (Ncf1<sup>*/*</sup>) mice. Synovial gene expression of NOX2-subunits was measured with qRT-PCR. Joint pathology was assessed using histology and antibodies against aggrecan neo-epitope VDIPEN. Levels of inflammatory proteins were measured with Luminex or ELISA. Phagocytes present in synovium, blood, bone marrow and spleen were analysed with flow cytometry. Extracellular ROS production by bone marrow-derived phagocytes was measured using an Amplex Red ROS detection assay. <h3>Results</h3> CiOA induction in knee joints of WT mice caused significantly increased synovial gene expression of NOX2 subunits. On day 7 of CiOA, synovial thickening, synovial S100A8/A9 levels and synovial percentages of inflammatory macrophages, PMNs, and monocytes were comparable between WT and Ncf1<sup>*/*</sup> mice. Cartilage damage and MMP activity, as measured by VDIPEN staining, were comparable, as well as levels of inflammatory mediators in serum and phagocyte percentages in blood, bone marrow and spleen. On day 42 of CiOA, synovitis, cartilage damage, and osteophyte formation in Ncf1<sup>*/*</sup> mice were unaltered when compared to WT mice. ROS production by Ncf1<sup>*/*</sup> PMNs was completely absent but Ncf1<sup>*/*</sup> macrophages, the more predominant phagocyte involved in development of pathology during CiOA, produced ROS in similar amounts as WT macrophages. Ncf1 deficiency thus seems to exclusively affect PMNs, this surprising finding might explain the lack of differences observed between CiOA development in WT and Ncf1<sup>*/*</sup> mice. ROS production by WT and Ncf1<sup>*/*</sup> macrophages was strongly upregulated by S100A8 and almost completely inhibited by the pan-NOX inhibitor diphenyleneiodonium chloride (DPI). In order to determine whether NOX1 complexes caused the compensation responsible for Ncf1 independent ROS production, we co-stimulated Ncf1<sup>*/*</sup> macrophages with NOX1-specific inhibitor ML171. However, no ML171-induced inhibition of ROS production was observed. <h3>Conclusions</h3> Absence of PMN-derived ROS does not alter synovitis and joint pathology in S100A8/A9-driven experimental inflammatory OA. The mechanism that enables Ncf1-independent ROS production by macrophages should be further investigated. <h3>Disclosure of Interest</h3> None declared