THU0046 Calcium pyrophosphate and monosodium urate crystal-induced prostaglandin E2 production involves NF-κB activation and ros production, independently of INTERLEUKIN-1BETA axis
Authors: F Renaudin, L Campillo-Gimenez, C Combes, M Gosset, M Cohen-Solal, F Lioté, H-K Ea
Journal: Annals of the Rheumatic Diseases (2017)

<h3>Background</h3> Monoclinic and triclinic calcium pyrophosphate dihydrated (mCPPD and tCPPD) and monosodium urate (MSU) crystals are responsible in human for relapsing acute arthritis. CPP and MSU crystal-triggered inflammation depends on several inflammatory mediators including interleukin (IL)-1β and prostaglandin (Pg) E2. IL-1β production is governed by NF-κB, NLRP3 inflammasome and caspase-1 activation. PgE<sub>2</sub> derives from arachidonic acid (AA) synthesis, which is regulated by cytosolic phospholipase A2 (cPLA<sub>2</sub>), and cyclooxygenase-(COX) 2 activation. CPP crystal-induced IL-1β production is well documented, but CPP crystal-induced PgE2 production remains unclear. <h3>Objectives</h3> To evaluate how CPP crystals induce PgE<sub>2</sub> production and the role of IL-1β in this process. <h3>Methods</h3> Synthetic and pyrogen-free m-CPPD, t-CPPD and MSU crystals were used to stimulate human monocyte cell line (THP-1 cells) and primary bone marrow-derived macrophages (BMDM) of wild type (wt) and NLRP3 inflammasome deficient (nlrp3<sup>-/-</sup>) mice. Pharmacological inhibitors were used to assess the role of oxidative stress (N-acetyl-L-cysteine, NAC) and NF-κB pathway (Bay-117085). PgE<sub>2</sub> and IL-1β production were quantified by ELISA, gene expression by qRT-PCR, cPLA<sub>2</sub> by immunoblot. NF-κB activation was assessed in THP-1 cells containing a reporter gene under control of NF-κB p65 promotor. <i>In vivo</i>, CPP crystal-induced PgE2 production was evaluated in the air pouch model in the presence or not of NF-κB inhibitor or NAC. <h3>Results</h3> <i>In vitro</i>, m- and t-CPPD and MSU crystals rapidly induced the production of PgE<sub>2</sub> and IL-1β by THP-1 cells and BMDM. PgE<sub>2</sub> production was associated with cPLA<sub>2</sub>and NF-κB activation along with increased expression of COX-2 (20 fold) and its receptor EP2 and EP4 genes. While CPP crystal-induced IL-1β production was abolished in THP-1 cells by treatment with caspase-1 inhibitor and in nlrp3<sup>-/-</sup> BMDM, CPP crystal-induced PgE<sub>2</sub> was not modified suggesting IL-1β- and NLRP3-independent pathways. Interestingly, CPP crystal-induced PgE<sub>2</sub> production was completely abrogated by NF-κB inhibitor treatment and significantly decreased by the antioxidant NAC; in both case, COX-2 gene expression was dramatically inhibited. <i>In vivo</i>, treatment with NAC or Bay strongly inhibited IL-1β and PgE<sub>2</sub> production and cellular infiltrate induced by CPP crystals. <h3>Conclusions</h3> PgE<sub>2</sub> production mediated by CPP and MSU crystals is activated by NF-κB signaling, independently of NLRP3/IL-1β production axis. Moreover, ROS production, known as a NLRP3 activator in response to CPPD or MSU crystals, is also involved in the regulation of PgE<sub>2</sub> production. The relations between these different pathways are under investigation. <h3>Disclosure of Interest</h3> None declared