Effect of reactive inflammation in osteoarthritis on extracellular traps formation by circulating neutrophils

Abstract

The results of recent studies demonstrating the presence of pro-inflammatory mediators in osteoarthritis (OA), as well as known similarity of histological signs of inflammation in rheumatoid arthritis (RA) and OA, suggest an important role of NETosis in immune inflammation in OA patients. Our objective was to assess ability of blood neutrophils from ОA patients to generate NETosis spontaneously and after in vitro induction, and impact of reactive synovitis on the dynamics of NETosis indexes. Thirty-one patients with verified OA and 30 healthy volunteers were included into the study. Circulating neutrophils were isolated with one-step density gradient centrifugation using double layers of Iohexol gradient. Subpopulational profile of isolated neutrophil fractions, their viability, and nonspecific activation were evaluated microscopically using Trypan Blue exclusion test, as well as nitro-blue tetrazolium test. NETs were induced by phorbol-12-myristate13-acetate (PMA). Spontaneous and induced formation of NETs was assessed using fluorescence microscopy. The ОA patients were in clinical remission at the time of inclusion in the study. In 23 OA patients, an exacerbation was diagnosed during the study. The neutrophil fractions showed high purity and a high content of viable nonactivated cells. These parameters were comparable in the study groups. Mean percentage of spontaneous NETs in OA patients in remission was significantly increased comparing to healthy controls. Usage of PMA, as inducing agent was accompanied by a significant increase in ability of neutrophils to form NETs. Transition of OA to exacerbation was characterized by further significant increase in spontaneous and PMA-induced NETs. Spontaneous and induced NETs in OA patients at acute stage of the disease are significantly higher than in OA patients in remission state. The growth rate of spontaneous NET formation is 3.74 times higher than the induced NET formation in OA patients upon exacerbation. Statistically significant increase in the ability of peripheral neutrophils to spontaneous and induced formation of extracellular traps was found, depending on the stage of osteoarthritis. The data obtained suggest an opportunity for participation of circulating neutrophils via NETosis in pathogenesis of immune inflammation in OA.