Netosis in patients with systemic lupus erythematosus

Abstract

The article presents the data on assessment of functional features of neutrophils in 34 patients with systemic lupus erythematosus (SLE). Development of neutrophil extracellular traps (NETs) was evaluated in cell cultures incubated in vitro for 30 and 150 minutes (basal levels, NETBAS30 and NETBAS150, respectively), and in the presence of heat-inactivated S. аureus (strain ATCC 25923, 108 CFU/ml) (stimulated levels, NETST30 and NETST150, respectively). NET looks like thin free-lying extracellular fibrillar structures, 2-3 times exceeding the size of unchanged granulocyte. The result was expressed as percentage and relative amount of extracellular traps per 100 counted leukocytes. Phagocytic activity of neutrophils was evaluated as phagocytosis of S. аureus by counting the percentage of neutrophils that engulfed phagocytic index of microbial particles (PI); the average number of phagocytosed objects per neutrophil phagocytic number (PC). ROS-producing activity was determined in the reduction of Nitroblue Tetrazolium tested in spontaneous and stimulated S. аureus variants (NBTBAS and NBTST, respectively). The result was expressed as the percentage of formazan-positive cells per 100 white blood cells. Nitroxide-producing properties were determined using the Crow (1999) method in spontaneous and stimulated samples for the accumulation of the nitrated amino acid tyrosine (3-nitrothyrosine, 3-NTBAS, and 3-NTST, respectively). We revealed a decrease in ROS production, phagocytosis and NO-forming activity of neutrophils associated with increased netosis. Activation of the netosis was observed in cell cultures without stimulation, indicating the in vivo formation of networks in SLE. The NET increase is most pronounced in the patients with lupus nephritis (p < 0.05), and in remission of the disease (p < 0.05). We have revealed a correlation of NET formation parameters with duration and degree of SLE activity (rs = -0.6; p = 0.001, and rs = 0.39; p = 0.02, respectively); autoantibody titers (anti-dsDNA and ANA) (rs = 0.67; р = 0.047 and rs = 0.59; р = 0.034, respectively); prothrombin complex activity (rs = 0.6; p = 0.036), as well and urea and creatinine levels (rs = 0.47; p = 0.037 and rs = 0.39; p = 0.048, respectively). The parameters of NETs can be considered a promising biomarker for verifying the diagnosis of SLE, evaluation of clinical activity, disease severity, and predicting the development of complications.