Neutrophil extracellular trap formation and nuclease activity in septic patients

Abstract 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 Blood was withdrawn and NET formation from neutrophils in vitro was quantified (average percentage of neutrophils showing NET formation on an image) without stimulation and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.