Superoxide induces Neutrophil Extracellular Trap Formation in a TLR-4 and NOX-dependent mechanism.

Ahmed B Al-Khafaji,Allan Tsung,Hai Huang,Hamza Obaid Yazdani,Samer Tohme,David Miller

doi:10.2119/molmed.2016.00054

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.

Highlights

Superoxide released into circulation serves to activate neutrophils to produce proinflammatory cytokines, [25] yet it is unknown whether superoxide stimulates neutrophils to form neutrophil extracellular traps (NETs)
When neutrophils form NETs, decondensed chromatin studded with various proteins is expelled to form distinctive extracellular web-like structures. [3,4] We visualized this process by immunofluorescence after treatment of purified neutrophils in culture with superoxide
We found that superoxide treatment of Toll-like receptor 4 (TLR-4) KO neutrophils resulted in significantly less MPO-DNA complexes released compared with WT and approached the amount observed with media treatment alone (Figure 3C)

Summary

INTRODUCTION

NETs have been implicated as harmful contributors to the pathogenesis of methicillin-resistant Staphylococcus aureus bacteremia [6] and noninfectious inflammatory conditions including venous thrombosis, [7] transfusion-related acute lung injury, [8] cancer [9] and various autoimmune conditions. [5]. Pathologic oxidative stress after liver I/R has been attributed to ROS superoxide (O2-), generated by transfer of an electron to molecular oxygen. Mechanisms of superoxide generation include aberrant electron leak across the mitochondrial respiratory chain [17] and electron transfer from NADPH to molecular oxygen by NADPH oxidase (NOX). [25] Activation of traditional neutrophil mechanisms in the noninfectious setting of liver I/R produces sterile inflammation that exacerbates tissue injury by generating additional ROS and releasing inflammatory mediators and various proteolytic enzymes. Our study sought to determine whether superoxide can induce NET formation, to resolve the underlying molecular signaling cascade by which this occurs, and to determine the effect of antisuperoxide therapy on NET formation in a model of liver I/R. Our study reveals an association between acute oxidative stress after I/R established by superoxide and pathophysiologic immune response by NETs

MATERIALS AND METHODS

RESULTS

CONCLUSION