Effects of the antioxidants Trolox, Tiron and Tempol on neutrophil extracellular trap formation

• Antioxidant Trolox, a water-soluble analog of α-tocopherol, and nitroxide Tempol reduce PMA-induced NETosis of human neutrophils in a dose-dependent manner.
• Antioxidant Tiron does not interfere NETosis of human neutrophils under a broad range of concentrations.
• Trolox, Tempol and Tiron suppress PMA-induced ROS release significantly and in a dose-dependent manner.
• Antioxidants Trolox and Tempol can be recommended for treating autoimmune and inflammatory diseases associated with ROS-dependent NET release.

Neutrophils can entrap and kill pathogens by releasing of neutrophil extracellular traps (NETs), in addition to their routine functions such as phagocytosis and degranulation. NETs consist of a DNA backbone supplemented by multiple bactericidal proteins from the nucleus, the cytoplasm and the granules. Neutrophils release NETs after their activation by a number of physiological and pharmacological stimuli. In addition to the antimicrobial function, NETs are involved in the pathogenesis of various autoimmune and inflammatory diseases. Since NET formation predominantly depends on the generation of reactive oxygen species (ROS), all substances that are capable of scavenging ROS or inhibiting the enzymes responsible for their synthesis should prevent ROS-associated NET release. The aim of this study was to test substances with an antioxidant activity, such as Trolox, Tiron, and Tempol, for their capacity to inhibit NET formation by primary human neutrophils in vitro. We revealed for the first time an inhibitory effect of Trolox on ROS-dependent NET release. We also established a suppressive effect of Tempol on NET formation that manifested itself in a wide range of concentrations. In this study, no inhibitory influence of Tiron on NET release was revealed. All tested substances exerted a significant dose-dependent antioxidative effect on ROS generation induced by phorbol 12-myristate 13-acetate (PMA). We suggest that the antioxidants Trolox and Tempol should be recommended for treating autoimmune and inflammatory diseases that implicate ROS-dependent NET release.