Background Calcium pyrophosphate (CPP) crystal-induced inflammation is mainly driven by interleukin (IL)-1β. IL-1β production involves a two-step process including the formation of pro-IL-1β through NF-κB activation and its maturation inti active IL-1β through NLRP3 inflammasome activation. Two CPP crystal phases, namely monoclinic and triclinic CPP dehydrated (m- and t-CPPD) crystals are identified in synovial fluids. We have recently demonstrated that m- and t-CPPD display different inflammatory potentials marked by a differential level of IL-1β production and other pro-inflammatory mediator expression.
Objectives We aimed to assess the intracellular pathways upstream CPP crystal-induced IL-1β production, according to the different inflammatory properties of CPP crystal phases.
Methods Four synthesized and pyrogen-free CPP crystals [a-CPP (amorphous), m-CPPD or tetrahydrated (m-CPPT) and t-CPPD] (Gras P et al. 2014) and monosodium urate (MSU) crystals were used, in vitro, to stimulate THP-1 cell line or mouse bone marrow-derived macrophages (BMDM) in the presence or not of pharmacological inhibitors: Bay-117085 (Bay, NF-κB inhibitor); SB203580 (SB, MAPK p38 inhibitor); SP600125 (SP, JNK inhibitor) and PD98059 (PD, p42/44 MAPK inhibitor). The expression of pro- and anti-inflammatory cytokine genes was assessed by qRT-PCR, and IL-1β or IL-8 production by ELISA; MAPK phosphorylation by immunoblot. NF-κB activation was quantified in THP-1 cells containing a gene reporter plasmid under control of NF-κB transcriptional factor. In vivo, we used murine air pouch model to assess the effects of NF-κB inhibition in CPP crystal-mediated inflammation.
Results We previously demonstrated that in vitro m-CPPD crystals were the most inflammatory CPP crystals and induced a higher production of mature IL-1b associated to a higher expression of NLRP3 protein, and a higher IL-8 production and expression of inflammatory cytokine genes (IL-1β, IL-6, IL-8, IL-33, TNF, COX-2) than t-CPPD, m-CPPT and MSU crystals. a-CPP crystals did not have inflammatory property. m-CPPD crystals induced a higher NF-kB activation and a higher phosphorylation p38, p42/44 and JNK MAPKs than t-CPPD and MSU crystals. In parallel, we showed that inhibition of NF-κB completely abrogated mature IL-1b and IL-8 secretion and pro-IL-1β synthesis induced by all CPP crystals. Inhibition of JNK and p42/44 MAPK with SP and PD respectively, decreased both IL-1β secretion and NF-κB activation induced by CPP crystals. In vivo IL-1β and IL-8 production and neutrophil infiltration induced by m-CPPD crystals were dramatically decreased by NF-κB inhibitor.
Conclusions Our results showed that IL-1β and IL-8 production induced by CPP crystals is completely dependent of NF-κB signaling. They suggested that the inflammatory potential of different CPP crystals relied on their capacity to activate both MAPK and NF-κB pathways. Differential level of NF-κB activation is partially dependent of MAPK phosphorylation. Additional studies are ongoing to investigate the underlying mechanisms.
Gras P et al Structure of the calcium pyrophosphate monohydrate phase (Ca2P2O7.H2O): towards understanding the dehydration process in calcium pyrophosphate hydrates. Acta Crystallogr C Struct Chem. 2014.
Disclosure of Interest None declared