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Cited by 188Neutrophil, Myeloperoxidase and Oxidative MechanismsInflammation biomarkers and pathwaysImmune Response and Inflammation

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Mitochondrial DNA neutrophil extracellular traps are formed after trauma and subsequent surgery

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McIlroy, Daniel J., Jarnicki, Andrew G., Au, Gough G., Lott, Natalie, Smith, Doug W., Hansbro, Philip M., Balogh, Zsolt J.·2014·Journal of Critical Care
DOI 10.1016/j.jcrc.2014.07.013
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Abstract

INTRODUCTION: Neutrophil extracellular traps (NETs) have not been demonstrated after trauma and subsequent surgery. Neutrophil extracellular traps are formed from pure mitochondrial DNA (mtDNA) under certain conditions, which is potently proinflammatory. We hypothesized that injury and orthopedic trauma surgery would induce NET production with mtDNA as a structural component. METHODS: Neutrophils were isolated 8 trauma patients requiring orthopedic surgery postinjury and up to 5 days postoperatively. Four healthy volunteers provided positive and negative controls. Total hip replacement patients acted as an uninjured surgical control group. Neutrophil extracellular traps were visualized with DNA (Hoechst 33342TM/Sytox Green/MitoSox/MitoTracker) stains using live cell fluorescence microscopy with downstream quantitative polymerase chain reaction analysis of DNA composition. RESULTS: Neutrophil extracellular traps were present after injury in all 8 trauma patients. They persisted for 5 days postoperatively. Delayed surgery resulted in NET resolution, but they reformed postoperatively. Total hip replacement patients developed NETs postoperatively, which resolved by day 5. Quantitative polymerase chain reaction analysis of NET-DNA composition revealed that NETs formed after injury and surgery were made of mtDNA with no detectable nuclear DNA component. CONCLUSIONS: Neutrophil extracellular traps formed after major trauma and subsequent surgery contain mtDNA and represent a novel marker of heightened innate immune activation. They could be considered when timing surgery after trauma to prevent systemic NET-induced inflammatory complications.

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Crossref · 10.1016/j.jcrc.2014.07.013

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Fig. 1 Viable neutrophil nuclear DNA was stained with membrane permeable Hoechst 33342 (blue), and NETs were visualized by membrane impermeable Sytox Green (green) staining extracellular DNA. Negative control, healthy volunteer without stimulation (A) and positive control, healthy volunteer stimulated with 600 nmol/L PMA for 45 minutes (B).
Fig. 1 Viable neutrophil nuclear DNA was stained with membrane permeable Hoechst 33342 (blue), and NETs were visualized by membrane impermeable Sytox Green (green) staining extracellular DNA. Negative control, healthy volunteer without stimulation (A) and positive control, healthy volunteer stimulated with 600 nmol/L PMA for 45 minutes (B).CC-BY-NC-ND · From the PDF
Fig. 2 Viable neutrophil nuclear DNA was stained with membrane permeable Hoechst 33342 (blue), and NETs were visualized by membrane impermeable Sytox Green (green) staining extracellular DNA. Acetabular fracture patient, immediately postinjury (A); 7 days postinjury, immediately preoperative (B); immediately postoperative (C); and 5 days postoperative (D).
Fig. 2 Viable neutrophil nuclear DNA was stained with membrane permeable Hoechst 33342 (blue), and NETs were visualized by membrane impermeable Sytox Green (green) staining extracellular DNA. Acetabular fracture patient, immediately postinjury (A); 7 days postinjury, immediately preoperative (B); immediately postoperative (C); and 5 days postoperative (D).CC-BY-NC-ND · From the PDF
Fig. 3 Viable neutrophil nDNA was stained with membrane permeable Hoechst 33342 (blue), and NETs were visualized by membrane impermeable Sytox Green (green) staining extracellular DNA. Total hip replacement patient immediately preoperative (A), immediately postoperative (B), 3 days (C), and 5 days postoperative (D).
Fig. 3 Viable neutrophil nDNA was stained with membrane permeable Hoechst 33342 (blue), and NETs were visualized by membrane impermeable Sytox Green (green) staining extracellular DNA. Total hip replacement patient immediately preoperative (A), immediately postoperative (B), 3 days (C), and 5 days postoperative (D).CC-BY-NC-ND · From the PDF
Fig. 4 Neutrophil extracellular traps isolated from a pelvic fracture patient 5 days postoperative. Viable neutrophil nDNA was stained with membrane permeable Hoechst 33342 (blue), and NETs were visualized by membrane impermeable Sytox Green (green) staining extracellular DNA. Extracellullar mtDNA was stained with MitoSox [4] (red). Intracellular mitochondria are labeled with MitoTracker (red) (scale bar, 10 μ m). Neutrophil extracellular trap stained with MitoSox (A), NET stained with Sytox Green (B). C, Overlaid image: viable neutrophils (blue, Hoechst), colocalized mtDNA-NET (yellow, Sytox Green + MitoSox Red), and intracellular mitochondria (red/pink, Mitotracker).
Fig. 4 Neutrophil extracellular traps isolated from a pelvic fracture patient 5 days postoperative. Viable neutrophil nDNA was stained with membrane permeable Hoechst 33342 (blue), and NETs were visualized by membrane impermeable Sytox Green (green) staining extracellular DNA. Extracellullar mtDNA was stained with MitoSox [4] (red). Intracellular mitochondria are labeled with MitoTracker (red) (scale bar, 10 μ m). Neutrophil extracellular trap stained with MitoSox (A), NET stained with Sytox Green (B). C, Overlaid image: viable neutrophils (blue, Hoechst), colocalized mtDNA-NET (yellow, Sytox Green + MitoSox Red), and intracellular mitochondria (red/pink, Mitotracker).CC-BY-NC-ND · From the PDF

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Metadata

Authors
McIlroy, D. J., Jarnicki, A. G., Au, G. G., Lott, N., Smith, D. W., Hansbro, P. M., & Balogh, Z. J.
Affiliations
  • University of Newcastle Australia (AU)
  • Hunter Medical Research Institute (AU)
  • John Hunter Hospital (AU)
Corresponding author
Balogh, Zsolt J.
ORCID
Venue
Journal of Critical Care
Publisher
Elsevier BV
Published
2014-12
Volume
29
Issue
6
Pages
1133.e1-1133.e5
ISSN
0883-9441 (ISSN-L)
DOI
10.1016/j.jcrc.2014.07.013
OpenAlex
W2100947278
Cited by
187
References
27
Citations by year
2026: 10 · 2025: 18 · 2024: 20 · 2023: 22 · 2022: 23 · 2021: 17 · 2020: 18 · 2019: 16 · 2018: 12 · 2017: 17 · 2016: 9 · 2015: 5
FWCI
4.70
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Critical Care (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Figshare (submittedVersion)
  • UTS ePRESS (University of Technology Sydney) (submittedVersion, other-oa)
  • NOVA (University of Newcastle Australia) (submittedVersion)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Neutrophil, Myeloperoxidase and Oxidative Mechanisms
  • Life Sciences › Immunology and Microbiology › Immunology › Inflammation biomarkers and pathways
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
Keywords
neutrophil extracellular traps, mitochondrial DNA, trauma (physical injury), orthopedic surgery, timing of surgery, total hip arthroplasty, innate immune activation, postoperative inflammation, neutrophils
MeSH terms
Adult, DNA, Mitochondrial, Humans, Male, Middle Aged, Neutrophils, Wounds and Injuries, Case-Control Studies, Bacteremia, Systemic Inflammatory Response Syndrome, Arthroplasty, Replacement, Hip, Fractures, Bone, Extracellular Traps

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