Superoxide induces Neutrophil Extracellular Trap Formation in a TLR-4 and NOX-Dependent Mechanism

Abstract

Neutrophils constitute the early innate immune response to perceived infectious and sterile threats. Neutrophil extracellular traps (NETs) are a novel mechanism to counter pathogenic invasion and sequelae of ischemia, including cell death and oxidative stress. Superoxide is a radical intermediate of oxygen metabolism produced by parenchymal and nonparenchymal hepatic cells, and is a hallmark of oxidative stress after liver ischemia-reperfusion (I/R). While extracellular superoxide recruits neutrophils to the liver and initiates sterile inflammatory injury, it is unknown whether superoxide induces the formation of NETs. We hypothesize that superoxide induces NET formation through a signaling cascade involving Toll-like receptor 4 (TLR-4) and neutrophil NADPH oxidase (NOX). We treated neutrophils with extracellular superoxide and observed NET DNA release, histone H3 citrullination and increased levels of MPO-DNA complexes occurring in a TLR-4-dependent manner. Inhibition of superoxide generation by allopurinol and inhibition of NOX by diphenyleneiodonium prevented NET formation. When mice were subjected to warm liver I/R, we found significant NET formation associated with liver necrosis and increased serum ALT in TLR-4 WT but not TLR-4 KO mice. To reduce circulating superoxide, we pretreated mice undergoing I/R with allopurinol and N-acetylcysteine, which resulted in decreased NETs and ameliorated liver injury. Our study demonstrates a requirement for TLR-4 and NOX in superoxide-induced NETs, and suggests involvement of superoxide-induced NETs in pathophysiologic settings.

Hydrophilic titanium surfaces reduce neutrophil inflammatory response and NETosis

“…14,15,26,29 NETosis was initially thought to be merely another facet of this antimicrobial role, as NETs are released in response to bacterialdependent Toll-like Receptor (TLR) or immunoglobulin-dependent Fc receptor activation. 17,30,31 However, NETs have since been shown to form under various sterile inflammatory conditions, including autoimmune disease, 32 crystallopathy, 22 and organ injury. 20,21,33 In sterile inflammation, NETs enhance the pro-inflammatory response through the cytotoxicity of free histones, 34 transformation of pro-IL-1β into active IL-1β by NET-based enzymes, 35 and direct action as a DAMP signal for other immune cells.…”

“…13,15,16 Neutrophil activation is often triggered by pathogen-associated molecular patterns (PAMPs) binding to toll-like receptor-4 (TLR-4) during non-sterile inflammation. 17 These events have been long understood to serve antimicrobial purposes: propagate the immune response, limit pathogen activity, and enhance clearance of bacteria. However, recent studies have shown a role for neutrophils-namely, NETosisin sterile inflammation, which may be triggered by release of danger-associated molecular patterns (DAMPs).…”

The definition of neutrophil extracellular traps and the concentration of short-chain fatty acids in salmonella-induced inflammation of the intestine against the background of vancomycin and Bacteroides fragilis

“…At present, the most studied model is suicidal netosis, which lasts for 2-4 hours. Its onset is characterized by neutrophil activation, through the recognition of pathogens, which leads to the induction of the NADPH oxidase (NOX) complex, through protein kinase C, and to an increase in cytosolic Ca + [21][22][23]. These cations serve as a co-factor for peptidyl-arginase deaminase 4 (PAD4) and a nuclear enzyme that deaminates histones, modifies amino acids, which leads to decondensation of chromatin and loss of positive charges necessary for the interaction of histones with DNA [24,25].…”

Metabolic abnormalities and oxidative stress in lupus

“…NETosis in LDGs is at least in part dependent on mitochondrial ROS production, as treatment with the mitochondrial ROS scavenger MitoTempo significantly abrogates the extrusion of NET structures in these cells [51]. Neutrophils exposed to extracellular superoxide also display enhanced NET formation and this is associated with concomitant liver injury [72]. Of consequence, the study by Lood et el.…”