Patients´ and Volunteers´ characteristics
We included 18 consecutive septic patients admitted to our intensive care unit (ICU), as well as 27 healthy volunteers, mostly hospital staff, to this prospective, observational trial. The study was registered retrospectively (German clinical trials database, DRKS no. 00007694) following local ethics committee approval (no. 09-4154). Septic patients were eligible if they fulfilled the criteria of sepsis according to the Surviving Sepsis Campaign Guidelines (14). Of note all patients fulfill the now valid Sepsis-3 criteria. Volunteers were eligible if they did not suffer from any acute or chronic disease, had no vaccination within 14 days prior to blood withdrawal, and did not take chronic medications (except for oral contraception pills in women). To exclude that volunteers had an unrecognized infection, white blood count and C-reactive protein concentrations were measured and found to be within the normal reference range. Patients’ and volunteers’ characteristics are presented in table 1.
Table 1: see at the end of file, table should appear here
Procedures and measurements
Within 24 hours after first diagnosing sepsis blood was withdrawn, neutrophils were isolated (see below), and NET-formation assays were performed immediately, both in septic patients (n=18) and healthy volunteers (n=27). For the measurement of serum nuclease activity blood was stored on ice upon withdrawal, centrifuged (2000 g for 10 min), and serum was frozen at -80 °C until analysis.
Isolation of neutrophils
Primary blood-derived neutrophils were isolated from fresh blood by density gradient centrifugation using Polymorphprep™ (Progen Biotechnik, Heidelberg, Germany), as described previously (15). For in vitro NET formation assays, the neutrophils were seeded on poly-L-lysine-coated glass slides in 24-well plates at a concentration of 5×105 cells per well (250 µl) in RPMI 1640 medium (Thermo Fisher Scientific Inc., Waltham, MA) at 37 °C and 5% CO2, and NET formation was analyzed after incubation for 2 and 4 hours, respectively.
Isolation of mitochondrial DNA
To harvest a high amount of pure mtDNA, the cultivated human cell line HepG2 was used, as described previously (16). The cultivation took place in RPMI medium mixed with 10% fetal calf serum (FKS) and 1mM sodium pyruvate (Thermo Fisher Scientific Inc., Waltham, MA) and cells were stored at 37 °C and 5% CO2. For the isolation of mitochondria, the Mitochondria Isolation Kit for Cultured Cells (Thermo Fisher Scientific Inc., Waltham, MA) was used according to product description. Purified mitochondria sediments were collected and stored at 4 °C for 24 hours before mtDNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The mtDNA concentration was determined photometrically at a wavelength of 280 nm (Biophotometer Plus, Eppendorf, Hamburg, Germany). mtDNA was stored at -20 °C.
Assessment and induction of NET formation
NET formation was assessed in septic patients (n=18) and healthy volunteers (n=27) without stimulation (baseline) and following incubation with mtDNA (final concentration: 10 µg/well), Furthermore, phorbol 12-myristate 13-acetate (PMA, 25 nM final concentration), an approved NET formation inductor (17), was used as positive control to ensure overall stimulability of neutrophils. In detail, 250 µl RPMI medium including either the respective stimulating agent or vehicle (negative control) was added to 250 µl of cell suspensions (5x105 cells/well) in 24 well plates.
To achieve adhesion of neutrophils to the glass-insert all plates were centrifuged at room temperature (22 °C) at 512 g. Incubation time was 2 and 4 hours at 37 °C and 5% CO2, respectively. Neutrophils and neutrophil derived structures were fixed with 150 µl of 16% paraformaldehyde and the plates were stored at 5 °C until immunostaining.
Visualization and quantification of NET formation
NET visualization and quantification was performed as described previously (15), In detail, cell preparations were washed three times with phosphate-buffered saline (PBS), and permeabilized by incubation with 2% bovine serum albumin (BSA) in 0.2% Triton X-100/PBS for 45 min at room temperature. A mouse monoclonal anti DNA/histone H1 complex (mouse IgG2a anti DNA/histone antibody, Merck Millipore, Darmstadt, Germany) was added and cells were incubated overnight at 4 °C. After washing the cells three times with PBS an Alexa-Fluor-488-labelled goat-anti-mouse antibody (Thermo Fisher Scientific Inc., Waltham, MA) was added for 45 min at room temperature. Cells were then washed again, and slides mounted in ProlongGold® antifade with 4',6-Diamidino-2-Phenylindole (DAPI) (Invitrogen, Carlsbad, CA) and NET formation was analyzed using fluorescence microscopy (Leica TCS SP5 confocal microscope and Zeiss Anxioveit 200M non-confocal fluorescence microscope). From each slide three images were randomly selected. Data are presented as a percentage of cells showing NET formation related to all neutrophils of an image. For statistical analysis, the mean value of 6 images was used for calculation of average values for each condition and individual.
Quantification of nuclease activity
Serum nuclease activity of the consecutive septic patients and healthy volunteers was quantified by gel electrophoresis. As negative control tris-(hydroxymethyl)-aminomethan (TRIS)-buffer (300 mM TRIS, 50 mM calcium chloride, 50 mM magnesium chloride) was used. A dilution series of the DNase I (Sigma Aldrich, St. Louis, MO), with an activity range from 2 to 0,0035 units/ml, served as a positive control. TRIS-buffer and calf thymus DNA (Sigma Aldrich, St. Louis, MO) in a concentration of 1 mg/ml was added to the serum samples as well as to the positive and the negative controls. Samples were incubated for 18 hours at 37 °C. A phenol chloroform (Carl Roth, Karlsruhe, Germany) extraction was used to separate the DNA from proteins. The resulting fluid phase was mixed with loading buffer (Thermo Fisher Scientific Inc., Waltham, MA) and added to an agarose gel pocket (1%), followed by gel electrophoresis at 100 V for 30 minutes.
For semiquantification of serum nuclease activity, we compared the gel lane from samples to a dilution series of DNase I. The possible activity range was related to categories 1-6. This grading correlates with DNase I activity of zero (activity range 1), <0,007 U/ml (range 2), 0,007 U/ml (range 3), 0,007-0,015 U/ml (range 4), 0,015-0,06 U/ml (range 5), and ≥0,06 U/ml (range 6), respectively.
Microsoft Excel 2016 (V16, Microsoft, Redmond, WA) and GraphPad Prism (V 6, GraphPad Software, San Diego, CA) were used for data analysis. Data are presented as means (± standard deviation) unless indicated otherwise. The Student’s two-tailed t-test for independent samples or, in case of violation of the normality assumption (as tested by the Kolmogorov-Smirnov and Shapiro-Wilk tests), the Wilcoxon signed rank test was used. Potential associations between serum nuclease activity and clinicopathogenic variables of septic patients like C-reactive protein and procalcitonin serum concentrations were determined using Spearman correlation analysis. Null hypotheses were rejected and statistical significance assumed with an a priori alpha error p of less than 0.05.