Effects of purinergic-receptor antagonism on neutrophil inflammatory response induced by calcium pyrophosphate dihydrate crystals

MARÍA BELÉN LUFFI; FLORENCIA FERNÁNDEZ BAUER; RODRIGO BLAS; VICTORIA BORGIA; ALFREDO BORGIA; MARÍA CRISTINA PISTORESI; MARÍA SILVIA DI GENARO; CAROLINA GORLINO

San Luis

Congreso; LXXI Reunión Anual de la Sociedad Argentina de Inmunología; 2023

Sociedad Argentina de Inmunología

Deposition of calcium pyrophosphate dihydrate (CPP) crystals in joints and softtissues is the cause of acute or chronic inflammatory responses known asCalcium Pyrophosphate Crystal Deposition Disease (CPPD). CPP crystals ariseextracellularly and their formation requires sufficient extracellular PPi, which isderived from local production by the breakdown of ATP. Purinergic P2Y receptorsrepresent a main class of receptors by which neutrophils respond to extracellularnucleotides and the drug suramin is widely used as a general inhibitor of P2Yreceptors. We have previously showed that the presence of CPP crystals insynovial fluid from CPPD patients induced in neutrophils extracellular DNAexposure as well as an up-regulation of CD66b expression and reactive oxygenspecies (ROS) generation. The aim of this study was to shed light on possibleroles of purinergic signaling on neutrophil pro-inflammatory response induced byCPP crystals. Purified human neutrophils, isolated from healthy subjects, weretreated with suramin (30 μM) and then incubated with CPP crystals (CPP).Degranulation (measured by the expression of CD66b), ROS production(determined by DHR-123 assay) and NET formation (detected by Sytox Greenfluorescence) was studied by flow cytometry in basal neutrophils (basal),neutrophils stimulated with CPP crystals (control) or in neutrophils treated withsuramin 1 h before CPP crystal-stimulation (suramin treatment). Results areexpressed as mean ± SEM and p value is the result of one-way ANOVA (KruskalWallis test) followed by Dunn’s post-hoc test. We found that suramin exhibited apartial inhibitory effect on extracellular DNA exposure in neutrophils which werestimulated with CPP crystals (%SYTOX-Green positive cells, control (43 ± 5%)vs suramin treatment (20 ± 8%); p=0.05; n=9). Moreover, treatment of humanneutrophils in vitro with suramin inhibited the respiratory burst induced by CPPcrystals (% control (112 ± 3%) vs % suramin treatment (88 ± 7%); p=0.02; n=10).No effects were observed in CD66b expression (Gm control (144 ± 16) vs Gmsuramin treatment (136 ± 8); p=0.8; n=5). In conclusion, we showed thatpurinergic receptor inhibitor suramin seems to reduce the pro-inflammatory actionof CPP crystals on neutrophils. Further studies are needed to determine whichpurinergic receptors are involved in this process.