Background/Purpose: Calcium pyrophosphate crystals including monoclinic and triclinic dihydrate phases (m- and t-CPPD) are found in 40% of end-stage osteoarthritis (OA) patients. Frequently asymptomatic, it can give rise to synovitis contributing to OA lesion worsening. Microcrystal inflammation is orchestrated by macrophage interleukin (IL)-1β secretion which required a 2-step process: synthesis of precursor form proIL-1β and its cleavage to mature IL-1β by active caspase 1 depending on the NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome activation. Several pathways can be entailed in inflammasome activation in response to danger signals: ATP-dependent potassium (K⁺) efflux, reactive oxygen species (ROS) production, or mitochondrial disruption. We have previously demonstrated that m- and t-CPPD differentially induced NLRP3 protein expression and IL-1β production but intracellular mechanism leading to NLRP3 inflammasome activation is not clearly defined.

Methods: Human THP-1 or THP-1 ρ° cells (treated with ethidium bromide), and bone marrow-derived macrophages (BMDM) from wild type (wt) or P2X7 receptor knock-out (p2x7r^-/-) mice were primed before stimulation with synthetic m- and t-CPPD crystals in presence or absence of K⁺-enriched media (KCl 50mM – to block K⁺ efflux), N-acetyl-L-cystein (NAC 50mM – an intracellular ROS scavenger) or oxidized ATP (oxATP 200µM – an antagonist of ATP receptor). IL-1β and extracellular ATP (ATP_e) concentrations were measured in cell culture supernatants whereas intracellular ROS production and mitochondrial membrane potential were evaluated using fluorescent probes (DFDA and JC-1, respectively).

Results: First, we observed that CPPD stimulation induced a de novoROS production combined with a stronger mitochondrial membrane depolarization following m-CPPD than t-CPPD crystal stimulation. This latter effect and IL-1β production were inhibited in presence of NAC. Moreover, we showed a drastic inhibition of IL-1β maturation process in THP-1 ρ° cells which have a mitochondrial ROS production deficiency. Second, K⁺-enriched media on one hand inhibited CPPD-mediated IL-1β production by THP-1 cells and on the other hand restored basal levels of ROS and mitochondrial membrane potential. Finally, we found that m- and t-CPPD crystals differentially brought on an ATP release and that IL-1β production was partially inhibited by oxATP. However, although ATP_e can trigger K⁺ efflux through P2X7 receptor opening, we observed a similar or only a weak decreased IL-1β production between wt and p2x7r^-/- BMDM after t-CPPD and m-CPPD stimulation respectively. In contrast, IL-1β production is completely abrogated in presence of KCl as well wt as p2x7r^-/- BMDM.

Conclusion: We demonstrated that K⁺ efflux triggered by CPPD crystals is the first signal leading to mitochondrial ROS production and disruption which are considered as intracellular danger signal required for NLRP3 activation and IL-1β production. Interestingly, K⁺ efflux is partially dependent on P2X7 receptor through ATP release but the main K⁺ channel has to be defined.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inflammasomeil-1%ce%b2-activation-induced-by-calcium-pyrophosphate-dihydrate-crystals-is-mainly-driven-by-a-p2x7-receptor-independent-potassium-efflux/