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Background: There is little knowledge, whether in patients with sepsis neutrophil extracellular trap (NET) formation and NET degrading nuclease activity are altered. Thus, we tested the hypotheses that 1) NET formation from neutrophils of septic patients is increased compared to healthy volunteers, both without stimulation and following incubation with mitochondrial DNA (mtDNA), a damage-associated molecular pattern, or phorbol 12-myristate 13-acetate (PMA; positive control); and 2) serum nuclease activities are increased as well.
Methods: We included 18 septic patients and 27 volunteers in this prospective observational trial while study was registered retrospectively. Blood was withdrawn and NET formation from neutrophils in vitro was quantified (average percentage of neutrophils showing NET formation on an image) without stimulation and following incubation with mtDNA (10µg/well) or PMA (25nmol). Serum nuclease activity was assessed using gel electrophoresis.
Results: In contrast to our hypothesis, compared to healthy volunteers unstimulated NET release from neutrophils in septic patients was decreased by 46.3% (4.3%±1.8 SD vs. 8.2%±2.9, p≤0.0001) and 48.1% (4.9%±2.5 vs. 9.4%±5.2, p=0.002) after 2 and 4 hours of incubation. mtDNA further decreased NET formation in neutrophils from septic patients (4.7%±1.2 to 2.8%±0,8; p=0.03) but did not alter NET formation in neutrophils from volun-teers. As expected, PMA, as positive control, increased NET formation to 73.2% (±29.6) in septic patients and to 91.7% (±7.1) in volunteers after 4 hours of incubation (p=0.22). Serum nuclease activity (range: 0-6) was decreased in septic patients by 39.6% (3±2 vs 5±0; median and ICR, p=0.0001) compared to volunteers.
Conclusions: Unstimulated NET formation and nuclease activity are decreased in septic patients and mtDNA can further reduce NET formation. Thus, neutrophils from septic patients show decreased NET formation in vitro despite diminished nuclease activity in vivo. Trail registration DRKS00007694, German Clinical Trials database (DRKS). Registered retrospectively 06.02.2015.
Keywords
Neutrophil extracellular traps, sepsis, nuclease activity, mitochondrial DNA
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CURRENT STATUS: Published
View the published article at BMC Anesthesiology.
Version 2
Posted 19 Nov, 2019
No community comments so far
Editorial decision: Minor revision
On 29 Nov, 2019
Review #2 received
Received 26 Nov, 2019
Reviewer #1 agreed
On 14 Nov, 2019
Review #1 received
Received 14 Nov, 2019
Reviewer #2 agreed
On 14 Nov, 2019
Reviewers invited
Invitations sent on 13 Nov, 2019
Editor assigned
On 04 Nov, 2019
Submission checks complete
On 03 Nov, 2019
Editor invited
On 03 Nov, 2019
No comments provided
Editorial decision: Major revision
On 04 Sep, 2019
Review #2 received
Received 03 Sep, 2019
Review #1 received
Received 15 Aug, 2019
Reviewer #2 agreed
On 13 Aug, 2019
Reviewer #1 agreed
On 08 Aug, 2019
Reviewers invited
Invitations sent on 02 Aug, 2019
Submission checks complete
On 29 Jul, 2019
Editor assigned
On 16 Jul, 2019
Editor invited
On 15 Jul, 2019
First submitted
On 13 Jul, 2019
Metrics
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PDF Downloads: ...
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Subject Areas
Anesthesiology & Pain Medicine
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See the published version of this preprint at BMC Anesthesiology.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at BMC Anesthesiology.
Version 2
Posted 19 Nov, 2019
No community comments so far
Editorial decision: Minor revision
On 29 Nov, 2019
Review #2 received
Received 26 Nov, 2019
Reviewer #1 agreed
On 14 Nov, 2019
Review #1 received
Received 14 Nov, 2019
Reviewer #2 agreed
On 14 Nov, 2019
Reviewers invited
Invitations sent on 13 Nov, 2019
Editor assigned
On 04 Nov, 2019
Submission checks complete
On 03 Nov, 2019
Editor invited
On 03 Nov, 2019
No comments provided
Editorial decision: Major revision
On 04 Sep, 2019
Review #2 received
Received 03 Sep, 2019
Review #1 received
Received 15 Aug, 2019
Reviewer #2 agreed
On 13 Aug, 2019
Reviewer #1 agreed
On 08 Aug, 2019
Reviewers invited
Invitations sent on 02 Aug, 2019
Submission checks complete
On 29 Jul, 2019
Editor assigned
On 16 Jul, 2019
Editor invited
On 15 Jul, 2019
First submitted
On 13 Jul, 2019
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Anesthesiology & Pain Medicine
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at BMC Anesthesiology.
Background: There is little knowledge, whether in patients with sepsis neutrophil extracellular trap (NET) formation and NET degrading nuclease activity are altered. Thus, we tested the hypotheses that 1) NET formation from neutrophils of septic patients is increased compared to healthy volunteers, both without stimulation and following incubation with mitochondrial DNA (mtDNA), a damage-associated molecular pattern, or phorbol 12-myristate 13-acetate (PMA; positive control); and 2) serum nuclease activities are increased as well.
Methods: We included 18 septic patients and 27 volunteers in this prospective observational trial while study was registered retrospectively. Blood was withdrawn and NET formation from neutrophils in vitro was quantified (average percentage of neutrophils showing NET formation on an image) without stimulation and following incubation with mtDNA (10µg/well) or PMA (25nmol). Serum nuclease activity was assessed using gel electrophoresis.
Results: In contrast to our hypothesis, compared to healthy volunteers unstimulated NET release from neutrophils in septic patients was decreased by 46.3% (4.3%±1.8 SD vs. 8.2%±2.9, p≤0.0001) and 48.1% (4.9%±2.5 vs. 9.4%±5.2, p=0.002) after 2 and 4 hours of incubation. mtDNA further decreased NET formation in neutrophils from septic patients (4.7%±1.2 to 2.8%±0,8; p=0.03) but did not alter NET formation in neutrophils from volun-teers. As expected, PMA, as positive control, increased NET formation to 73.2% (±29.6) in septic patients and to 91.7% (±7.1) in volunteers after 4 hours of incubation (p=0.22). Serum nuclease activity (range: 0-6) was decreased in septic patients by 39.6% (3±2 vs 5±0; median and ICR, p=0.0001) compared to volunteers.
Conclusions: Unstimulated NET formation and nuclease activity are decreased in septic patients and mtDNA can further reduce NET formation. Thus, neutrophils from septic patients show decreased NET formation in vitro despite diminished nuclease activity in vivo. Trail registration DRKS00007694, German Clinical Trials database (DRKS). Registered retrospectively 06.02.2015.
Figure 1
Figure 2
Figure 3
Figure 4
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