INDUCTION OF NEUTROPHIL-DERIVED REACTIVE OXYGEN SPECIES AND NEUTROPHIL EXTRACELLU- LAR TRAP GENERATION BY CALCIUM PYROPHOSPHATE DIHYDRATE CRYSTALS

LUFFI, MARÍA BELÉN; BLAS, RODRIGO; BORGIA, VICTORIA; BORGIA, MARCOS; BORGIA, IVÁN; BORGIA, ALFREDO; PISTORESI, MARÍA CRISTINA; DI GENARO, MARÍA SILVIA; GORLINO, CAROLINA

Mar del Plata

Congreso; LXX REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INMUNOLOGÍA (SAI); 2022

Sociedad Argentina de Inmunología

Deposition of calcium pyrophosphate dihydrate (CPP) crystals in joints and soft tissues is the cause of acute or chronic inflammato- ry responses known as Calcium Pyrophosphate Crystal Deposition Disease (CPPD). Cell activation by CPP crystals is a central feature and pro-inflammatory crystals can interact with all of the major sy- novial cell types, including neutrophils. Although the neutrophil is a major inflammatory cell type found in inflamed joints of acute CPPD patients, little is known about interactions of neutrophils with CPP crystals. In this study, we aimed at studying whether CPP crystals induce a pro-inflammatory profile in neutrophils, measured by de- granulation, induction of reactive oxygen species (ROS) and neutro- phil extracellular trap (NET) generation. Neutrophils were isolated from healthy subjects and incubated with synthesized CPP crystals (CPP) or with synovial fluid from CPPD patients (CPPD-SF). De- granulation (measured by the expression of CD66b), ROS produc- tion (determined by DHR-123 assay) and NET formation (detected by Sytox Green fluorescence) was studied by flow cytometry in bas- al neutrophils (control), neutrophils stimulated with synthesized CPP crystals (CPP) or with CPPD-SF. P value is the result of one-way ANOVA (􏰇rus􏰈al-􏰉allis test) followed by Dunn􏰃s post-hoc test or Mann-Whitney test, when appropriate. We found that the presence of CPP crystals in SF from CPPD patients induced extracellular DNA exposure in neutrophils (%SYTOX-Green positive cells, control vs CPPD-SF; p=0.03) as well as an up-regulation of CD66b expres- sion (p=0.03). Moreover, CPPD-SF samples significantly increased respiratory burst in neutrophils (control vs CPPD-SF, p=0.04). In conclusion, these data suggest that the presence of CPP crystals in SF from CPPD patients could have a role in the amplification of the inflammatory response by stimulating up-regulation of CD66b, NET release and ROS production in neutrophils.