POS0389 NEUTROPHIL AND MONOCYTE EXTRACELLULAR TRAPS IN RHEUMATOID ARTHRITIS: POTENTIAL SOURCE OF DIAGNOSTIC BIOMARKERS

Abstract

Background:Up-to date quite high rheumatoid arthritis (RA) prevalence has also a trend towards steady increase for several decades. Earliest possible diagnosis and personalized treatment of RA are commonly believed to be principal way to prevent or diminish joint destruction. Emerging biomarkers retrieval has therefore critical importance for improvement of RA treatment outcomes, especially for its biological treatment. One of upcoming sources of new biomarkers is newly discovered phenomenon of neutrophil extracellular traps (NET) and monocyte extracellular traps (MET) formation, specifically in RA.Objectives:Evaluation of peripheral blood neutrophils and monocytes ability to generate NET and MET spontaneously and after induction in vitro in RA.Methods:The research was carried out in agreement with the WMA Declaration of Helsinki principles. 30 patients with verified RA according to the ACR/EULAR 2010 criteria were included in the study. 30 healthy volunteers were enrolled as a reference group. RA disease activity was assessed using DAS28 score did not exceeded 2.6 in every patient at the inclusion timepoint. Neutrophils and monocytes were isolated with one-step centrifugation procedure using three-layer ficoll-amidotrizoate density gradient with density of upper, intermediate and lower layers 1068 kg/m3, 1080 kg/m3, and 1090 kg/m3, respectively. The cell types in the resulting fractions were identified histochemically, and the extent of cell activation was assessed using common nitro-blue tetrazolium test. Generation of NETs was stimulated by phorbol-12-myristate-13-acetate (PMA). Generation of METs was stimulated using bacterial LPS. The shape and size of NETs and ETs were assessed using fluorescence microscopy with SYBR green.Results:Mean contamination fraction of neutrophils and monocytes in the reference group did not exceeded 3% and 2%, respectively. Mean purity of neutrophil fraction in RA group was 93.1±6.1%, and cell viability in every sample was above 95.4±5.1%. Mean purity of monocyte fraction in RA group was 98.4±6.4%, cell viability in every sample was above 93.4±4.8%. Spontaneous NET and MET formation was observed in neutrophils and monocytes isolated from both RA patients and, significantly less, in healthy controls. Neutrophils from ACPA-positive RA patients were found to reveal increased spontaneous and induced NETs formation compared to ACPA- negative RA patients. Monocytes did not demonstrate any difference between these subgroups.Conclusion:NETs could probably be considered as a candidate source of citrulline autoantigen participating in autoantibody production, whereas METs may play less important role in this phenomenon. NETs and ETs can be considered as potential diagnostic biomarkers of RA. Further studies of NETosis and ETosis in RA patients can promote emerging researches for targeted therapy of RA.Disclosure of Interests:None declared