Human neutrophils were isolated from peripheral blood of healthy donors using a dextran-
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1 Materials and Methods Isolation of neutrophils Human neutrophils were isolated from peripheral blood of healthy donors using a dextran- Ficoll method (S1). Transmission electron microscopy For fine structural analysis, cells were fixed with 2.5% glutaraldehyde, postfixed with 1% osmiumtetroxide, contrasted with uranylacetate and tannic acid, dehydrated and embedded in Polybed (Polyciences). After polymerization, specimens were cut at 60 nm and contrasted with lead citrate. For immunodetection, cells were fixed with 4% PFA and embedded in a mixture of 25% sucrose/10% PVA. Ultrathin sections were cut at 105 C, blocked, reacted with primary antibodies followed by secondary antibodies coupled to 6 or 12 nm gold particles. Specimens were analyzed in a Leo 906E transmission electron microscope. Scanning electron microscopy Cells were fixed with 2.5% glutaraldehyde, postfixed using repeated incubations with 1% osmium tetroxid / 1% tannic acid, dehydrated with a graded ethanol series, critical-point dried and coated with 2 nm platinum. Since the NETs are fragile, each step was done with minimal disturbance of the media to preserve the structures. For immunodetection, cells were fixed with 4% PFA, blocked, reacted with primary antibodies followed by secondary antibodies coupled to 12 nm gold particles. After dehydration and critical-point drying, the specimens were coated with 5 nm carbon and analyzed in a Leo 1550 scanning electron microscope.
2 Immunofluorescence assays Neutrophils were seeded on glass coverslips treated with 0.001% polylysine, allowed to settle and either treated with IL-8 (10-7 M, R&D Systems), PMA (25 nm) or LPS (100 ng/ml, List Biologicals) or left unstimulated. Cells were fixed with 4% PFA, blocked overnight (10% normal goat serum, 5% cold water fish gelatine, 1% bovine serum albumin, 0.05% Tween 20 in PBS) and incubated with primary antibodies which were detected with secondary antibodies coupled to Cy2 or Cy3. Controls were done with isotype matched controls. For DNA detection, DRAQ5 (shown) BisBenzimide (Hoechst 33342), Sytox, and ToPro3 were used. Specimens were analyzed with a Leica TCS-SP confocal microscope. Quantification of DNA release from activated neutrophils Freshly isolated neutrophils were seeded into 96-well plates and stimulated with PMA at concentrations between and 50 nm for 30 min (dose response) and with 10 nm PMA for 10, 20 and 30 min (time course). After the indicated time points, Sytox Green (Molecular Probes), a non cell-permeant DNA binding dye, was added to the cells at a final concentration of 5 µm to detect extracellular DNA. Non-stimulated neutrophils were used a control. The plates were read in a fluorescence microplate reader (Thermo Labsystems, Helsinki, Finland) with a filter setting of 485(excitation)/527(emission). Bacterial strains and growth conditions S. flexneri strain M90T and S. aureus strain RN6390 (provided by R. Novick) were grown to the exponential phase of growth with aeration in tryptic soy broth and brain heart infusion medium respectively. S. typhimurium strain SL1344 was grown overnight in Luria Bertani medium at 37 o C without aeration.
3 Extracellular bacterial killing with intact neutrophils Human neutrophils were resuspended at 2x10 6 /ml, and allowed to adhere to plastic plates in media containing IL-8. After 30 min incubation at 37 o C, the medium was carefully replaced with serum free culture medium, containing 2% heat inactivated pooled human serum with or without cytochalasin D (10µg/ml) and incubated further for 15 minutes before infection with bacteria. Cytochalasin D treatment did not affect NETs and this concentration was effective in blocking bacterial phagocytosis. To show that NETs were important in extracellular killing, samples were pre-incubated with 100 U/ml of RNase and proteinase free DNase 1 (Worthington) to destroy NETs or anti-h2a-h2b-dna complex (PR1-3, kindly provided by M. Monestier) or an IgG2a isotype control (15µg/ml) before the addition of bacteria (2x10 4 /ml). Samples were centrifuged at 700g for 10 min and incubated at 37 o C and 5% CO 2 for 30 minutes. Bacterial killing was measured as percentages of control values (bacteria incubated alone in media without neutrophils). In the absence of cytochalasin D, bacterial killing of S. flexneri and S. aureus ranged between 60-70% and % respectively. Bactericidal activity Bacteria (10 6 for M90T and SL1344 and 10 5 for strain RN6390) were incubated with purified H2A (Roche Diagnostics Corporation) in a total volume of 100µl for 30 min at 37 o C with shaking in Hanks Balanced Salts Solution (HBSS - ) buffered with 10mM HEPES ph7.4 and supplemented with 0.3% Casamino acids. Aliquots were plated in Luria broth agar for the determination of colony forming units (CFU). Histology Rabbit infections were performed as described (S2) and human appendicitis histological samples were obtained from surgical removal. Tissue samples were fixed in 10% formalin, dehydrated, embedded in paraffin, cut at 10 µm, rehydrated and stained with hematoxylin and
4 eosin. For immunostainings, rehydrated samples were subjected to antigen retrieval, incubated with primary antibodies which were detected with secondary antibodies coupled to Cy2 or Cy3. DNA was detected with DRAQ5.
5 Figure S1 Legend to Figure S1 DNA release from neutrophils activated with PMA is dose- and time-dependent. The formation of NETs was tested in activated neutrophils by staining extracellular DNA with a cell-impermeable dye and using a fluorometer. (S1A) PMA concentrations as low as 5 nm induce the release of DNA. (S1B) At a concentration of 10 nm PMA, DNA is extruded as early as 10 min after activation.
6 Figure S2 Legend to Figure S2 α-hemolysin, a virulence factor of Staphylococcus areus, is degraded by NETs. Activated neutrophils were infected with S. aureus, fixed ans stained with the DNA stain DRAQ5 or by immunofluorsecence with an anti-α-hemolysin antibody. α-hemolysin was dim in bacteria exposed to NETs (upper right). In the presence of the serine protease inhibitor SLPI, the staining for α-hemolysin is much stronger (bottom right).
7 Figure S3 Legend to Figure S3 Antimicrobial activity of histone 2A. Purified H2A was added to log phase cultures of Staphylococcus aureus, S. flexneri and S. typhimurium as described in Materials and Methods. A dramatic dose-dependent reduction of cfu was observed.
8 Table S1 Protein cellular localisation NET inclusion NE azurophilic granules + MPO azurophilic granules + cathepsin G azurophilic granules + BPI azurophilic granules + Lactoferrin specific granules + Gelatinase tertiary granules + H1 nucleus + H2A nucleus + H2B nucleus + H3 nucleus + H4 nucleus + CD 63 granule membrane - annexin I cytoplasma - β-catenin cytoplasma - α-tubulin cytoplasma - f-actin cortex - cytochrom C mitochondria - Major component: DNA Legend to Table S1 Immunofluorescence staining pattern of activated neutrophils. Granule and nuclear components are present in NETs, while cytoplasmic proteins are excluded.
9 Legend to Movie S1 DNA is a major constituent of NETs. Brief treatment with protease-free DNase leads to complete dismantling of the fibres. Legend to Movie S2 Time-lapse movie of isolated PMN before and after PMA stimulation. Every 5 sec one frame was taken, the time-lapse factor is 120. Unstimulated, the cells are highly motile but stay spheroid most of the time. After addition of PMA (small image shift after half of the movie), the cells flatten considerably but remain highly motile. The last frame was taken after addition of the cell impermeant DNA dye Sytox Green which stains NETs. In the area of NETs, no dead cells are visible. References S1. Y. Weinrauch, et al., Nature 417, 91 (2002). S2. J. Arondel, et al., Infect.Immun. 67, 6056 (1999).
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