european crystal network workshop

    Calcium pyrophosphate dihydrate crystals induce IL-1β production by monocytes through a potassium efflux-dependent pathway

    Monoclinic and triclinic calcium pyrophosphate dihydrate (m- and t-CPPD) crystals are observed in human joints and synovial fluids. Frequently asymptomatic, it can give rise to arthritis contributing to osteoarthritis lesion worsening. CPP-induced inflammation is orchestrated mainly by interleukin (IL)-1β whose secretion required a cytokine maturation process dependent of NLRP3 inflammasome activation. This intracellular protein complex can be stimulated by potassium (K+) efflux, reactive oxygen species (ROS) generation, lysosomal or mitochondrial alterations. The objectives were to identify intracellular pathways induced by CPPD crystals and leading to NLRP3 activation and IL-1β production.

    The effects of CPPD crystals were assessed in human THP-1 cell line and bone marrow-derived macrophages (BMDM) from wild type (wt) or P2X7 receptor (p2x7-/-) knock out mice. Cells were primed before stimulation with synthetic m- and t-CPPD crystals in presence or not 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). NLRP3 expression was determined by western blotting, IL-1β and extracellular ATP (ATPe) concentrations were measured in cell culture supernatants whereas ROS production and mitochondrial membrane potential were evaluated using fluorescent probes (DFDA and JC-1, respectively).

    m-CPPD crystals led to a higher NLRP3 expression than t-CPPD suggesting a differential modulation of NLRP3 activation pathways. First, we observed that CPPD-induced IL-1β secretion was completely abrogated when K+ efflux or intracellular ROS were inhibited. However, m-CPPD crystal stimulation induced a stronger mitochondrial membrane depolarization than t-CPPD crystal combined with a de novo ROS generation. These two latter effects were inhibited when K+ efflux was blocked. Second, we found that m- and t-CPPD crystals differentially brought on an ATP release and that IL-1β production was partially inhibited by oxATP. Although ATPe can elicit a K+ efflux through P2X7 receptor opening, crystal-mediated IL-1β production was similar between wt and p2x7r -/- BMDM.

    IL-1β production triggered by m- and t-CPPD crystals occurred through a modulation of ROS production and mitochondrial membrane disruption. Interestingly, K+ efflux, associated with ATP release, could be the initial signal of CPPD-induced IL-1β maturation, independently of P2X7 receptor involvement.

     

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