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Cited by 204Neutrophil, Myeloperoxidase and Oxidative MechanismsRedox biology and oxidative stressBlood disorders and treatments

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ROS induces NETosis by oxidizing DNA and initiating DNA repair

Open access
Azzouz, Dhia, Khan, Meraj A., Palaniyar, Nades·2021·Cell Death Discovery
DOI 10.1038/s41420-021-00491-3PMC PMC8128883
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Abstract

Reactive oxygen species (ROS) are essential for neutrophil extracellular trap (NET) formation or NETosis. Nevertheless, how ROS induces NETosis is unknown. Neutrophil activation induces excess ROS production and a meaningless genome-wide transcription to facilitate chromatin decondensation. Here we show that the induction of NADPH oxidase-dependent NETosis leads to extensive DNA damage, and the subsequent translocation of proliferating cell nuclear antigen (PCNA), a key DNA repair protein, stored in the cytoplasm into the nucleus. During the activation of NETosis (e.g., by phorbol myristate acetate, Escherichia coli LPS, Staphylococcus aureus (RN4220), or Pseudomonas aeruginosa ), preventing the DNA-repair-complex assembly leading to nick formation that decondenses chromatin causes the suppression of NETosis (e.g., by inhibitors to, or knockdown of, Apurinic endonuclease APE1, poly ADP ribose polymerase PARP, and DNA ligase). The remaining repair steps involving polymerase activity and PCNA interactions with DNA polymerases β/δ do not suppress agonist-induced NETosis. Therefore, excess ROS produced during neutrophil activation induces NETosis by inducing extensive DNA damage (e.g., oxidising guanine to 8-oxoguanine), and the subsequent DNA repair pathway, leading to chromatin decondensation.

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Crossref · 10.1038/s41420-021-00491-3

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Fig. 1 PMA- and LPS-treated neutrophils generate increased levels of 8-oxoG. A Neutrophils were treated with PMA (25 nM) or LPS (5 μg/ml) and incubated for 240 min. Cells were stained for DNA (DAPI, blue) and 8-oxoG (purple). The images are representative of three independent experiments. Scale bar, 10 μm. B-C 8-oxoG levels following treatment with media (-ve control), PMA (25 nM) or LPS (5 μg/ml) were measured using an in-cell ELISA. LPS- and PMA-treated cells had significantly greater 8-oxoG levels compared to media treated cells (n = 6; *p < 0.05).
Fig. 1 PMA- and LPS-treated neutrophils generate increased levels of 8-oxoG. A Neutrophils were treated with PMA (25 nM) or LPS (5 μg/ml) and incubated for 240 min. Cells were stained for DNA (DAPI, blue) and 8-oxoG (purple). The images are representative of three independent experiments. Scale bar, 10 μm. B-C 8-oxoG levels following treatment with media (-ve control), PMA (25 nM) or LPS (5 μg/ml) were measured using an in-cell ELISA. LPS- and PMA-treated cells had significantly greater 8-oxoG levels compared to media treated cells (n = 6; *p < 0.05).CC BY · PubMed Central
Fig. 2 PCNA translocates into the nucleus/perinuclear regions following the induction of NETosis with PMA or LPS, and eventually decorates the NET DNA. A Neutrophils were treated with PMA (25 nM) or LPS (5 μg/ml) and incubated for 90 or 30 min, respectively. Cells were stained for DNA (DAPI, blue) and PCNA (red). B Neutrophils were treated with PMA (25 nM) or LPS (5 μg/ml) and incubated for 240 min. Cells were stained for DNA (DAPI, blue), PCNA (red) and MPO (green). The images are representative of three independent experiments. Scale bar, 10 μm.
Fig. 2 PCNA translocates into the nucleus/perinuclear regions following the induction of NETosis with PMA or LPS, and eventually decorates the NET DNA. A Neutrophils were treated with PMA (25 nM) or LPS (5 μg/ml) and incubated for 90 or 30 min, respectively. Cells were stained for DNA (DAPI, blue) and PCNA (red). B Neutrophils were treated with PMA (25 nM) or LPS (5 μg/ml) and incubated for 240 min. Cells were stained for DNA (DAPI, blue), PCNA (red) and MPO (green). The images are representative of three independent experiments. Scale bar, 10 μm.CC BY · PubMed Central
Fig. 3 Inhibition of early steps, but not late steps, of DNA repair suppresses NETosis induced by PMA and LPS. A DNA release from neutrophils following media (-ve control) or PMA (25 nM) was measured using the SYTOX Green plate reader assay. Cells were preincubated with BER inhibitors (APE inh 1, CRT0044876 (125 μM); APE inh 2, APE1 Inhibitor III (50 μM); PARP1 inh 1, BSI201 (100 μM); PARP inh 2, PJ34 (50 μM); LIG inh, L189 (100 μM); Pol β inh, AM-TS23 (25 μM); PCNA:Pol δ inh, T2AA (25 μM) or Pol δ inh, Aphidicolin (50 μM)) for 1 h prior to PMA treatment (n = 3; *p < 0.05 compared to positive control). B Neutrophils were incubated with DNA repair inhibitors for 1 h then treated with PMA (25 nM), incubated for 240 min, and stained for DNA (DAPI, blue). The images are representative of three independent experiments. Scale bar, 10 μm. C Differentiated HL-60 neutrophils were pretreated with media, a scrambled siRNA, APE1, or PARP siRNA. Cells were then treated with PMA (150 nM) or LPS (15 μg/ml) for 6–8 h. NETosis levels were measured using the SYTOX Green plate reader assay. Western blots were used for verifying knockdown. The data are presented as mean ± SEM (n = 4; *p < 0.05 compared to the control with no inhibitors).
Fig. 3 Inhibition of early steps, but not late steps, of DNA repair suppresses NETosis induced by PMA and LPS. A DNA release from neutrophils following media (-ve control) or PMA (25 nM) was measured using the SYTOX Green plate reader assay. Cells were preincubated with BER inhibitors (APE inh 1, CRT0044876 (125 μM); APE inh 2, APE1 Inhibitor III (50 μM); PARP1 inh 1, BSI201 (100 μM); PARP inh 2, PJ34 (50 μM); LIG inh, L189 (100 μM); Pol β inh, AM-TS23 (25 μM); PCNA:Pol δ inh, T2AA (25 μM) or Pol δ inh, Aphidicolin (50 μM)) for 1 h prior to PMA treatment (n = 3; *p < 0.05 compared to positive control). B Neutrophils were incubated with DNA repair inhibitors for 1 h then treated with PMA (25 nM), incubated for 240 min, and stained for DNA (DAPI, blue). The images are representative of three independent experiments. Scale bar, 10 μm. C Differentiated HL-60 neutrophils were pretreated with media, a scrambled siRNA, APE1, or PARP siRNA. Cells were then treated with PMA (150 nM) or LPS (15 μg/ml) for 6–8 h. NETosis levels were measured using the SYTOX Green plate reader assay. Western blots were used for verifying knockdown. The data are presented as mean ± SEM (n = 4; *p < 0.05 compared to the control with no inhibitors).CC BY · PubMed Central
Fig. 4 Inhibition of early steps of BER suppresses NETosis induced by P. aeruginosa and S. aureus. A, C DNA release from neutrophils following media (-ve control), 20 MOI P. aeruginosa or 20 MOI S. aureus (RN4220) was measured using the SYTOX Green plate reader assay. Cells were preincubated with BER inhibitors (APE inh 1, CRT0044876 (125 μM); APE inh 2, APE1 Inhibitor III (50 μM); PARP1 inh 1, BSI201 (100 μM); PARP inh 12, PJ34 (50 μM) or LIG inh, L189 (100 μM)) for 1 h prior to bacterial treatment. The data are presented a mean ± SEM (n = 3; *p < 0.05 compared to the control with no inhibitors). B Neutrophils were incubated with DNA repair inhibitors, that inhibited NETosis, for 1 h then treated with 20 MOI P. aeruginosa and incubated for 240 min, and stained for DNA (DAPI, blue). The images are representative of three independent experiments. Scale bar, 10 μm.
Fig. 4 Inhibition of early steps of BER suppresses NETosis induced by P. aeruginosa and S. aureus. A, C DNA release from neutrophils following media (-ve control), 20 MOI P. aeruginosa or 20 MOI S. aureus (RN4220) was measured using the SYTOX Green plate reader assay. Cells were preincubated with BER inhibitors (APE inh 1, CRT0044876 (125 μM); APE inh 2, APE1 Inhibitor III (50 μM); PARP1 inh 1, BSI201 (100 μM); PARP inh 12, PJ34 (50 μM) or LIG inh, L189 (100 μM)) for 1 h prior to bacterial treatment. The data are presented a mean ± SEM (n = 3; *p < 0.05 compared to the control with no inhibitors). B Neutrophils were incubated with DNA repair inhibitors, that inhibited NETosis, for 1 h then treated with 20 MOI P. aeruginosa and incubated for 240 min, and stained for DNA (DAPI, blue). The images are representative of three independent experiments. Scale bar, 10 μm.CC BY · PubMed Central
Fig. 5 Summary figure. Diagram showing the role of DNA damage repair during NETosis in the context of the other known components of the NETosis pathway.
Fig. 5 Summary figure. Diagram showing the role of DNA damage repair during NETosis in the context of the other known components of the NETosis pathway.CC BY · PubMed Central

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Metadata

Authors
Azzouz, D., Khan, M. A., & Palaniyar, N.
Affiliations
  • University of Toronto (CA)
  • Hospital for Sick Children (CA)
Corresponding author
Palaniyar, Nades
ORCID
Palaniyar, Nades
Venue
Cell Death Discovery
Publisher
Springer Science and Business Media LLC
Published
2021-05-18
Volume
7
Issue
1
Pages
113-113
ISSN
2058-7716 (ISSN-L)
DOI
10.1038/s41420-021-00491-3
PMC
PMC8128883
OpenAlex
W3163218484
Cited by
202
References
35
Citations by year
2026: 37 · 2025: 61 · 2024: 50 · 2023: 22 · 2022: 28 · 2021: 4
FWCI
9.89
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Death Discovery (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Cystic Fibrosis Canada (3180)
  • Gouvernement du Canada | Natural Sciences and Engineering Research Council of Canada (RGPIN436250-13)
  • Gouvernement du Canada | Canadian Institutes of Health Research (MOP-111012)
  • Hospital for Sick Children
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Neutrophil, Myeloperoxidase and Oxidative Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
  • Health Sciences › Medicine › Hematology › Blood disorders and treatments
Keywords
neutrophil extracellular traps, NETosis, reactive oxygen species, NADPH oxidase, DNA damage, DNA repair, chromatin decondensation, 8-oxoguanine, APE1, poly(ADP-ribose) polymerase, DNA ligase, neutrophil activation, DNA polymerase beta, DNA polymerase delta

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