SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx

Abstract

Neutrophils cast neutrophil extracellular traps (NETs) to defend the host against invading pathogens. Although effective against microbial pathogens, a growing body of literature now suggests that NETs have negative impacts on many inflammatory and autoimmune diseases. Identifying mechanisms that regulate the process termed “NETosis” is important for treating these diseases. Although two major types of NETosis have been described to date, mechanisms regulating these forms of cell death are not clearly established. NADPH oxidase 2 (NOX2) generates large amounts of reactive oxygen species (ROS), which is essential for NOX-dependent NETosis. However, major regulators of NOX-independent NETosis are largely unknown. Here we show that calcium activated NOX-independent NETosis is fast and mediated by a calcium-activated small conductance potassium (SK) channel member SK3 and mitochondrial ROS. Although mitochondrial ROS is needed for NOX-independent NETosis, it is not important for NOX-dependent NETosis. We further demonstrate that the activation of the calcium-activated potassium channel is sufficient to induce NOX-independent NETosis. Unlike NOX-dependent NETosis, NOX-independent NETosis is accompanied by a substantially lower level of activation of ERK and moderate level of activation of Akt, whereas the activation of p38 is similar in both pathways. ERK activation is essential for the NOX-dependent pathway, whereas its activation is not essential for the NOX-independent pathway. Despite the differential activation, both NOX-dependent and -independent NETosis require Akt activity. Collectively, this study highlights key differences in these two major NETosis pathways and provides an insight into previously unknown mechanisms for NOX-independent NETosis.

Signal Inhibitory Receptor on Leukocytes-1 Limits the Formation of Neutrophil Extracellular Traps, but Preserves Intracellular Bacterial Killing

“…This cellular process requires the generation of ROS (38, 42, 53). However, ROS-mediated signaling is not the only way that NET release is triggered, as rapid NET formation (within minutes) was described, which is independent of oxidants (41,54). Our present study shows that S. aureus triggers the formation of NETs through a mechanism that does not depend on ROS.…”

Histones as Mediators of Host Defense, Inflammation and Thrombosis

“…ROS: Reactive oxygen species. that NETs can be formed by more than one process [71][72][73][74]. The 'classic' mechanism of NET formation involves death of the neutrophil (recently termed 'suicidal NETosis').…”

Evasion of Neutrophil Extracellular Traps by Respiratory Pathogens

“…A second, NADPH oxidaseindependent, calcium-mediated NETosis pathway has also been described, involving the enzyme, protein arginine deiminase 4 (PAD4) (4). Upon the increase of intracellular calcium in response to agents such as calcium ionophores, PAD4 binds calcium, crosses into the nucleus, and deiminates positively charged arginine residues to generate uncharged citrullin on histones H3 and H4.…”

“…This loss of positive charge leads to the dissociation of the histones from negatively charged DNA, resulting in decondensation of the nucleosome. Citrullination of histones has been shown to be involved in calcium-dependent, but not NADPH oxidase-dependent, NETosis (4). It was demonstrated that PAD4 deficiency or use of Cl-amidine to chemically inhibit PAD4 activity causes a reduction of histone decondensation and NET formation (5).…”