Session Type: ACR Poster Session C
Session Time: 9:00AM-11:00AM
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 (ATPe) 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 ATPe 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.
To cite this abstract in AMA style:Campillo-Gimenez L, Renaudin F, Bobé P, Gosset M, Combes C, Cohen-Solal M, Lioté F, Ea HK. Inflammasome/IL-1β Activation Induced By Calcium Pyrophosphate Dihydrate Crystals Is Mainly Driven By a P2X7 Receptor-Independent Potassium Efflux [abstract]. Arthritis Rheumatol. 2016; 68 (suppl 10). 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/. Accessed July 10, 2020.
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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/