AB0073 NEUTROPHIL EXTRACELLULAR TRAPS: COUPLING WITH RHEUMATOID ARTHRITIS FLARES AND ANTI-CITRULLINE AUTOANTIBODIES PERSISTENCE

Abstract

BackgroundRheumatoid arthritis (RA) is characterized by dysregulation of the adaptive immune system. Detection of subcellular structures with localization of most typical citrulline-containing rheumatoid autoantigens in a single compartment is of special interest considering importance of anticitrulline autoantibodies for autoimmune response in RA. Neutrophil extracellular traps (NETs) can be suggested as a most likely candidate for the role of such structures.ObjectivesTo study of peripheral blood neutrophils from RA to generate NETs, associated with diseases exacerbation.MethodsThe research was carried out in agreement with the WMA Declaration of Helsinki principles after the local ethical board approval. Circulating neutrophils were isolated with one-step density gradient centrifugation using double layers of ficoll-amidotrizoate gradient. Composition of isolated cellular fractions, their viability, and non-specific activation were evaluated by light microscopy using common Romanowsky-Giemsa staining, trypan blue exclusion test as well as NBT test. Neutrophil fractions contained minute percentages of impurities and low extents of activated and dead cells. NETs were induced by PMA. Spontaneous and induced formation of extracellular traps was assessed using fluorescence microscopy [1]. Results were presented as values (95%CI).Results37 patients (6 males and 31 females, mean age 42.7 years) with verified RA according to the ACR/EULAR 2010 criteria were included in the study. RA disease activity at the inclusion timepoint shouldn’t exceed 2.6 DAS28 points. 16 (15.4%) patients had subsequent increase of the DAS28 score above 3.2 at following visits (3, 8 and 12 months after the inclusion). 30 healthy volunteers (9 males and 21 females, mean age 37.2 years) were enrolled as a reference group. Mean proportions of spontaneous and induced NET formation by isolated neutrophils of inactive RA patients (DAS 28<2.6) were 6.0 (5.7-6.3)% and 26.9 (24.0-29.8)%, respectively. Active RA patients (DAS 28>3.2) had dramatic increase of the respective values: 16,6 (16,4-16,8)% and 38,0 (37,7-38,3)%.Both active and inactive RA patients had significant increase of NET-generating neutrophils proportion comparing to the reference group as well as significant difference of both these parameters between active and inactive phase (р<0.05). The growth rate of spontaneous NET formation in the active RA was 183.8%, the respective value for induced NET formation was 46.7%. The growth rate of spontaneous NET formation is 3.9 times higher than the induced NET formation.Neutrophils from ACPA-positive RA patients were found to have increased spontaneous and induced NETs formation compared to ACPA-negative RA patients (р>0.05).ConclusionThus, our study demonstrated a strong relationship between onset of RA flare and increase of spontaneous and induced NET formation by circulating neutrophils. There was also a clear distinction between ACPA negative and positive RA patients as to higher spontaneous and induced NET formation in the latter subgroup. The comparison using larger group of patients will magnify the differences above significance limit. Continued research in this direction can promote the development of new therapies of RA.