The role of the mitochondrial pore in the effector functions of human neutrophils

• Vorobjeva N.V.
• Kondratenko I.V.
• Vakhlyarskaya S.S.
• Chernyak B.V.
• Boris V. Pinegin

Abstract

Introduction. Neutrophils release neutrophil extracellular traps (NETs) in response to numerous pathogenic microbes in the inflammatory foci. However, excessive NETosis or diminished NET clearance is engaged in the pathogenesis of numerous autoimmune and inflammatory disorders. The initiation of NETosis after recognition of pathogens by specific receptors is mediated by an increase in the intracellular Ca²⁺ concentration, therefore, the use of Ca²⁺ ionophore A23187 can be considered as semi-physiological model of NETosis. Induction of NETosis by various stimuli depends on reactive oxygen species (ROS) produced by enzymatic complex NADPH oxidase, however, NETosis induced by Ca²⁺ ionophores was suggested to be mediated by ROS produced in mitochondria (mtROS) or without any ROS in general.

Material and methods. A wide inhibitor analysis has been used in our work, as well as luminol-amplified chemiluminescence assay and fluorescence detection of neutrophil extracellular traps.

Results. Using mitochondria-targeted antioxidant SkQ1, we confirmed that mtROS are involved in the oxidative burst and NET formation activated by A23187, but not a powerful activator of protein kinase C PMA. At the same time, using specific inhibitors of NADPH oxidase, we showed that ROS produced by this enzymatic complex are also involved in NETosis induced by A23187. Application of specific inhibitors of mitochondrial pore (mPTP), allowed us to demonstrate for the first time that mPTP is involved in signal transduction from mitochondria to NADPH oxidase, in mtROS production, NETosis and the oxidative burst induced byA23187.

Conclusion. We assume that the interplay between two sources of ROS, NADPH oxidase and mitochondria, is triggered by Ca²⁺ influx, so that mtROS contribute to NETosis either directly or by stimulating NADPH oxidase. One of the possible physiological mechanisms mediating Ca²⁺-dependent increase in mtROS production may be associated with the opening of the mitochondrial permeability transition pore mPTP, a multicomponent protein complex located in the mitochondrial membrane. Excessive NETs formation or a decrease in the organism’s ability to eliminate them can cause the development of autoimmune diseases. NETs are also involved in the development of lung diseases. Therefore, understanding the signaling pathways of NET formation is extremely important for finding ways to combat these diseases.

Keywords:human neutrophils; oxidative burst; reactive oxygen species; neutrophil extracellular traps; mitochondrial permeability transition pore; mPTP

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