title: 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 non-parenchymal 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.
authors.affiliation: Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.
authors.forename: Ahmed B
authors.initials: AB
authors.lastname: Al-Khafaji
authors.affiliation: Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.
authors.forename: Samer
authors.initials: S
authors.lastname: Tohme
authors.affiliation: Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.
authors.forename: Hamza Obaid
authors.initials: HO
authors.lastname: Yazdani
authors.affiliation: Department of Internal Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA.
authors.forename: David
authors.initials: D
authors.lastname: Miller
authors.affiliation: Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.|Department of Surgery, Union Hospital, Huazhong University of Science and Technology, Wuhan, P.R. China.
authors.forename: Hai
authors.initials: H
authors.lastname: Huang
authors.affiliation: Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.
authors.forename: Allan
authors.initials: A
authors.lastname: Tsung
keywords: inflammation; ischemia; liver; oxidants; reactive oxygen species