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 production is governed by NF-κB, NLRP3 inflammasome and 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-κB p65 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 cPLA2and 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.
Conclusions 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.
Disclosure of Interest None declared
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