Pseudogout-Associated Inflammatory Calcium Pyrophosphate Dihydrate Microcrystals Induce Formation of Neutrophil Extracellular Traps

Pseudogout is an autoinflammatory condition triggered by calcium pyrophosphate dehydrate (CPPD) crystal deposition in the joints. The innate immune system is irritated by and responds to the presence of the crystals with an inflammatory response. The synovial fluid contains activated inflammatory macrophages and neutrophil granulocytes. Several details of crystalinduced macrophage activation were recently uncovered, but very little is known about interactions of CPPD crystals with neutrophils. In this study, we show that human neutrophils engulf CPPD crystals and form large amounts of neutrophil extracellular traps (NETs) in vitro. Released extracellular DNA binds myeloperoxidase and citrullinated histone H4. CPPD crystal-stimulated neutrophils and their nuclear DNA undergo morphological changes characteristic for NET formation. The ERK/MEK signaling pathway, heat shock protein 90, PI3K, and an intact cytoskeleton are required for CPPD-induced NET formation. Blocking crystal-activated respiratory burst has, however, no effect on NETs. Human neutrophils release IL-1 beta and IL-8 in response to CPPD crystals, and blocking CXCR2, the main IL-8R, diminishes NET formation. Proinflammatory cytokines, TNF-alpha, GM-CSF, and IL-1 beta, increase NET release by the crystals. Enhanced bacterial killing by CPPD-induced NETs demonstrates their ability to cause cellular damage. Our work documents and provides details about extracellular trap release in human neutrophils activated by CPPD microcrystals. We suggest that crystal-triggered NET formation can be a novel contributor to inflammatory conditions observed in CPPD crystal-driven synovitis.