Lipopolysaccharide-induced neutrophil extracellular trap formation in canine neutrophils is dependent on histone H3 citrullination by peptidylarginine deiminase

Abstract

Neutrophils release neutrophil extracellular traps (NETs), which are extracellular chromatin decorated with histones and antimicrobial proteins. Although known for antimicrobial properties, overzealous production of NETs (NETosis) may lead to cytotoxicity and multiple organ failure in sepsis. Pathogen-induced NETosis has been extensively studied in mice but its importance in dogs remains largely unknown. This study sought to characterize in vitro NETosis induced by E.coli LPS, including assessing the role of peptidylarginine deiminase (PAD) in canine NETosis. Neutrophils (1×10 cells/ml) from healthy dogs were isolated and treated with 100μg/ml LPS, 100nM phorbol 12-myristate 13-acetate (PMA), or buffer for either 90 or 180min. NETs were assessed using fluorescence microscopy of living neutrophils and immunofluorescent microscopy. Supernatant and cellular debris were purified and cell-free DNA was quantified by spectrophotometry. The role of PAD was assessed by treating LPS- and PMA-activated neutrophils with 50, 100 or 200μM of the PAD inhibitor, Cl-amidine. In vitro NETosis was characterized by co-localization of cell-free DNA, citrullinated histone H3, and myeloperoxidase. LPS stimulation resulted in intracellular citrullination of histone H3. Compared to PMA chemically-induced NETosis, LPS resulted in smaller NETs with less extracellular citrullinated histone H3. Cl-amidine decreased citrullination of histones and NET production in either LPS- or PMA-stimulated neutrophils demonstrating that neutrophil PAD is essential for these cellular processes.

Entamoeba histolytica Trophozoites Induce a Rapid Non-classical NETosis Mechanism Independent of NOX2-Derived Reactive Oxygen Species and PAD4 Activity

“…Because of its activity, the presence of citrullinated proteins, principally histones, is considered as a marker of PAD4-mediated NETosis (Brinkmann et al, 2016 ; Li et al, 2017 ). In accordance to other reports, our results showed that citrullinated proteins were scarce in control and PMA-treated neutrophils (Neeli and Radic, 2013 ; Konig and Andrade, 2016 ; Muth et al, 2017 ); however, abundant citrullinated proteins were seen in neutrophils treated with A23187 and amoebas.…”

A novel approach to identifying and quantifying neutrophil extracellular trap formation in septic dogs using immunofluorescence microscopy

“…Histone citrullination, catalzyed by the enzyme, peptidylarginine deiminase 4 (PAD4), alters the electrostatic interactions between DNA and histones causing chromatin decondensation and release of cfDNA during NETosis [ 18 ]. We have recently shown that lipopolysaccharide-mediated NETosis in vitro requires histone H3 hypercitrullination by PAD in canine neutrophils [ 9 ]. The substantial number of citH3 positive cells identified in culture-positive samples suggests that a similar cellular process also occurs in vivo.…”

“…To date, NETs have not been documented in dogs with bacterial sepsis. Isolated canine neutrophils have been shown to release NETs in response to E. coli lipopolysaccharide and the chemical, phorbol 12-myristate 13-acetate [ 8 , 9 ]. Several methods of plasma cfDNA quantification have been described in dogs with sepsis, neoplasia and immune-mediated haemolytic anemia [ 8 , 10 ].…”

Root extracellular traps versus neutrophil extracellular traps in host defence, a case of functional convergence?

“…ET formation was described for the first time in human neutrophils in 2004 (Brinkmann et al , ), but has since also been reported for several different phagocytic cells in mammal (e.g. dogs, rodents, horses) (Etulain et al , ; Li, Ng & Tablin, ; Vargas et al , ) and non‐mammal vertebrate species (e.g. crocodile, fish, chicken) (Palić et al , ; Kozlowski et al , ; Pieper et al , ; Wen et al , ), as well as for invertebrates such as the social amoeba Dictyostelium discoideum (Zhang et al , ) and the earthworm Eisenia andrei (Homa et al , ).…”