Félix Renaudin (1), Laure Campillo-Gimenez (1), Christèle Combes (2), Marjolaine Gosset (3), Martine Cohen-Solal (1,4), Frédéric Lioté (1,4), Hang-Korng Ea (1,4)
Affiliation(s):
1. INSERM UMR1132, Bioscar, University Paris Diderot, Paris,
2. CIRIMAT, Université de Toulouse, INPT,UPS, CNRS, ENSIACET, Toulouse,
3. EA 2496, UFR Odontologie, Paris Descartes, Montrouge,
4. AP-HP, hôpital Lariboisière, service de rhumatologie, centre Viggo Petersen, Paris
Background: 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 productionis governed by NF-κB, NLRP3 inflammasomeand caspase-1 activation. PgE2 derives from arachidonic acid (AA) synthesis, which is regulated by cytosolic phospholipase A2 (cPLA2), and cyclooxygenase-(COX) 2 activation. CPP crystal-induced IL-1β production is well documented, but CPP crystal-induced PgE2 production remains unclear.
Objectives: To evaluate how CPP crystals induce PgE2 production and the role of IL-1β in this process.
Methods: 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-/-)mice. Pharmacological inhibitors were used to assess the role of oxidative stress (N-acetyl-L-cysteine, NAC) and NF-κB pathway (Bay-117085). PgE2 and IL-1β production were quantified by ELISA, gene expression by qRT-PCR, cPLA2 by immunoblot. NF-κB activation was assessed in THP-1 cells containing a reporter gene under control of NF-κBp65 promotor. In vivo, CPP crystal-induced PgE2 production was evaluated in the air pouch model in the presence or not of NF-κB inhibitor or NAC.
Results: In vitro, m-and t-CPPD and MSU crystals rapidly induced the production of PgE2 and IL-1β by THP-1 cells and BMDM. PgE2 production was associated with cPLA2 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-/-BMDM, CPP crystal-induced PgE2 was not modified suggesting IL-1β- and NLRP3-independent pathways. Interestingly, CPP crystal-induced PgE2 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. In vivo, treatment with NAC or Bay strongly inhibited IL-1β and PgE2 production and cellular infiltrate induced by CPP crystals.
Conclusion: PgE2 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 PgE2 production. The relations between these different pathways are under investigation.