Laure Campillo-Gimenez1, Pierre Gras2, Christèle Combes2, Martine Cohen-Solal1,3, Frédéric Lioté1,3, Hang-Korng Ea1,3.
1INSERM UMR1132, Paris, France. 2ENSIACET, CIRIMAT, INPT-UPS-CNRS, Toulouse, France. 3 AP-HP, Lariboisière Hospital, Rheumatology Department, Paris, France.
Abstract: Interleukin-1β (IL-1β) plays a pivotal role in CPP crystal-induced inflammatory process but mechanisms of CPP crystal-mediated IL-1β production remain unknown. One possible pathway could imply the reactive oxygen species (ROS) reported as a cellular mediator of inflammasome/IL-1β activation. Four different types of CPP crystals are described: amorphous (a-CPP), monoclinic dihydrate or tetrahydrate (m-CPPD; m-CCPT) and triclinic dihydrate (t-CPPD). Only m-CPPD and t- CPPD crystals have been identified in human samples and the effect of different types of CPP crystals on IL-1β production has never been explored. Thus, the aim of the project was to evaluate the inflammatory properties of these 4 types of CPP crystals and elucidate the cellular pathways involved in IL-1β production. CPP crystals were synthesized as previously described (Gras P et al, Acta Cryst. 2014). Monosodium urate crystals (MSU) were used as positive control. In vitro, mature THP-1 cells (human monocyte cell line) were stimulated with CPP or MSU crystals (200 μg/ml) in the presence or not of N-acetyl-L-cysteine (NAC), an antioxidant molecule. At different times of culture, IL-1β production and expression of IL-1β, IL-6, TNF-α, IL-8, COX-2 or IL-10 genes were quantified. In vivo CPP crystal-mediated inflammation was assessed using the air pouch model in WT mice (n=6/groups). IL-1β production and cell infiltrate or IL-1β gene expression were evaluated in air pouchlavages and -membranes, respectively. In vitro m-CPPD crystals induced a higher IL-1β release than t-CPPD or MSU while a-CPP and m-CPPT crystals did not induce it. IL-1β production in response to crystals significantly increased from 2 to 48 hrs of stimulation and the presence of NAC significantly inhibited m-CPPD-mediated IL-1β production. Similarly, m-CPPD and t-CPPD crystals increased the pro-inflammatory gene expression with a maximum effect at 24 hrs of stimulation. Interestingly, maximum of IL-10 gene induction occurred at 6 hrs of stimulation. In vivo studies confirmed the increase in IL-1β production and gene expression associated with neutrophil and monocyte infiltration into the air pouch after CPP crystal injection. Diverse forms of CPP crystals displayed differential cellular responses, m-CPPD representing the most inflammatory CPP crystals in vitro. Preliminary results highlighted a role of ROS production in CPP crystal-induced IL-1β production. Investigations of their sources and mechanisms of generation are ongoing