Background/Purpose:

Monoclinic and triclinic calcium pyrophosphate (m- and t-CPP) crystals are the 2 types of CPP crystals observed in human joints. Frequently asymptomatic, it can give rise to acute arthritis. Interleukin (IL-) 1β plays a pivotal role in CPP crystal-induced inflammation but how they induce IL-1β production remains unknown. IL-1β secretion occurs after its maturation by caspase-1 which is activated by NLRP3 inflammasome. NLRP3 inflammasome can be stimulated by potassium (K⁺) efflux, reactive oxygen species (ROS) generation, lysosomal or mitochondrial alterations. In this study we aimed to decipher how CPP crystals initiate IL-1β production.

Methods:

Four types of CPP crystals were synthesized in vitro: m- and t-CPP dihydrate (m- and t-CPPD), amorphous CPP (a-CPP) and m-CPP tetrathydrate (m-CPPT) [1]. The effects of CPP and monosodium urate (MSU) crystals – the later used as control – were assessed in human THP-1 cell line and bone marrow-derived monocytes (BMDM) from wild type (wt) and P2X7 receptor knock-out (P2X7^-/-) mice. Cells were primed before stimulation with crystals. In vivo, m- and t-CPPD crystal effects were evaluated in the murine air pouch model. 

IL-1β production was measured in cell culture supernatants and air pouch lavages by ELISA. Expression of inflammatory mediator genes was analyzed by qPCR. ROS production and mitochondrial membrane potential were evaluated by microscopy and flow cytometry, using fluorescent probes (CellRox® and JC-1, respectively). Extracellular ATP (ATP_e) concentration was quantified in cell culture supernatants.

Results:

In vitro, we showed that m-, t-CPPD and MSU crystals differentially induced IL-1β secretion and IL-1β, IL-6, IL-8, TNF-α, COX2 gene expression (m-CPPD > MSU > t-CPPD) while a-CPP and m-CPPTβ crystals had no effect; IL-1β production was not correlated with CPP crystal specific surface area. Similarly, these 3 inflammatory crystals induced in vivo IL-1β secretion, neutrophil and monocyte influx into the air pouch. Then, we assessed the mechanisms of CPP crystal-induced IL-1β production. First, we found that m- and t-CPPD crystals brought on an ATP release and that IL-1β production was partially inhibited by oxidized ATP. Second, m- or t-CPPD-induced IL-1β secretion was completely abrogated when K⁺ efflux was inhibited (cell culture with a K⁺-enriched medium). Although ATP_e can trigger K⁺ efflux through P2X7 receptor opening, crystal-mediated IL-1β production was identical between wt and P2X7^-/-BMDM. Finally, we demonstrated an early decrease in mitochondrial membrane potential following crystal stimulation combined with a higher intracellular ROS level. Moreover, ROS scavenger dramatically decreased IL-1β release (75% inhibition) induced by CPPD crystals.

Conclusion:

CPP crystals displayed different inflammatory properties, m-CPPD crystals being the most potent one. CPPD crystal-induced IL-1β maturation occurs through three major mechanisms including ATP release, P2X7 receptor-independent K⁺efflux, and mitochondrial disruption/ROS production.

[1] Gras P. et al, Eur. J. Inorg. Chem., 2013

ACR Meeting Abstracts - https://acrabstracts.org/abstract/four-types-of-calcium-pyrophosphate-crystals-differentially-induce-il-1beta-production-by-monocytes-through-atppotassium-effluxros-dependent-pathways/