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) that serum nuclease activities are increased as well.

Methods: Following ethic committee approval, we included 18 septic patients and 27 volunteers in this prospective observational trial. Blood was withdrawn and NET formation from neutrophils was analyzed in vitro without stimulation and following incubation with mtDNA (10 μg/well) or PMA (25 nmol). Furthermore, serum nuclease activity was assessed using gel electrophoresis.

Results: In contrast to our hypothesis, in septic patients, unstimulated NET release from neutrophils 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 h compared to volunteers. 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 volunteers. Of note, using PMA, as positive control, we ensured that neutrophils were still able to form NETs, with NET formation increasing to 73.2% (±29.6) in septic patients and 91.7% (±7.1) in volunteers (p = 0.22). Additionally, we show that 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. mtDNA can further reduce NET formation in sepsis. Thus, neutrophils from septic patients show decreased NET formation in vitro despite diminished nuclease activity in vivo.

Trial registration: DRKS00007694, german clinical trials database (DRKS). Retrospectively registered 06.02.2015.

Keywords: Mitochondrial DNA; Neutrophil extracellular traps; Nuclease activity; Sepsis.

Fig. 1

Extracellular Trap (NET) formation by neutrophils obtained from blood of septic patients and volunteers following incubation for 2 and 4 h. Neutrophils from septic patients released significantly less NETs than those from volunteers under baseline conditions both after incubation for 2 and 4 h, respectively. Data are means ± SD

Fig. 2

Neutrophil Extracellular Trap (NET) formation in vitro at baseline (open symbols) and after 4 h of incubation with mitochondrial DNA (mtDNA, full symbols) in neutrophils from volunteers (left panel, A) and septic patients (right panel, B). While NET formation in neutrophils from volunteers (A) is unaltered by mtDNA, mtDNA evoked a marginal decrease in NET formation in neutrophils from septic patients (B). Data are individual values and means ± SD

Fig. 3

Neutrophil Extracellular Trap (NET) formation following phorbol-myristate-acetate (PMA) stimulation. PMA (25 nM) led to a strong increase of NET formation, both in septic patients and volunteers, reaching NET formation in more than 70% of neutrophils. Thus, in spite of decreased NET formation under baseline conditions in septic patients, NET formation capacity in neutrophils from septic patients is still high and approaches that of PMA stimulated neutrophils from volunteers. Data are means ± SD

Fig. 4

Nuclease activity in serum of patients with sepsis and volunteers. Patients with sepsis show a marked decrease of nuclease activity compared to that of volunteers. Data are median ± interquartile range