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 × 106 cells/ml) from healthy dogs were isolated and treated with 100 μg/ml LPS, 100 nM phorbol 12-myristate 13-acetate (PMA), or buffer for either 90 or 180 min. 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 200 μM 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 29-37 |
| Number of pages | 9 |
| Journal | Veterinary Immunology and Immunopathology |
| Volume | 193-194 |
| DOIs | |
| State | Published - Dec 1 2017 |
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Keywords
- Cell-free DNA
- Citrullinated histone
- Peptidylarginine deiminase
- Sepsis
ASJC Scopus subject areas
- Immunology
- veterinary(all)
Cite this
Lipopolysaccharide-induced neutrophil extracellular trap formation in canine neutrophils is dependent on histone H3 citrullination by peptidylarginine deiminase. / Li, Ronald H L; Ng, Geena; Tablin, Fern.
In: Veterinary Immunology and Immunopathology, Vol. 193-194, 01.12.2017, p. 29-37.Research output: Contribution to journal › Article
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TY - JOUR
T1 - Lipopolysaccharide-induced neutrophil extracellular trap formation in canine neutrophils is dependent on histone H3 citrullination by peptidylarginine deiminase
AU - Li, Ronald H L
AU - Ng, Geena
AU - Tablin, Fern
PY - 2017/12/1
Y1 - 2017/12/1
N2 - 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 × 106 cells/ml) from healthy dogs were isolated and treated with 100 μg/ml LPS, 100 nM phorbol 12-myristate 13-acetate (PMA), or buffer for either 90 or 180 min. 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 200 μM 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.
AB - 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 × 106 cells/ml) from healthy dogs were isolated and treated with 100 μg/ml LPS, 100 nM phorbol 12-myristate 13-acetate (PMA), or buffer for either 90 or 180 min. 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 200 μM 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.
KW - Cell-free DNA
KW - Citrullinated histone
KW - Peptidylarginine deiminase
KW - Sepsis
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UR - http://www.scopus.com/inward/citedby.url?scp=85032009833&partnerID=8YFLogxK
U2 - 10.1016/j.vetimm.2017.10.002
DO - 10.1016/j.vetimm.2017.10.002
M3 - Article
VL - 193-194
SP - 29
EP - 37
JO - Veterinary Immunology and Immunopathology
JF - Veterinary Immunology and Immunopathology
SN - 0165-2427
ER -