BCG strain and culturing
BCG Connaught substrain (ATCC35733) was from the American Type Culture Collection (Manassas, VA). BCG suspension was prepared in Middlebrook 7H9 broth (BD 271310) media and colonies cultured on 7H10 solid media (BD 262710, Difco Laboratories, USA). The details of medium were divided into solution A: Middlebrook 7H9 broth (0.47g), glycerin (0.2ml), tween80 (0.2ml), and solution B: bovine serum albumin (BSA) (0.5g), glucose (0.2g), NaCl (0.08g). Solution A+B was mixed into 100ml deionized water and was used to culture BCG. Viable BCG from the logarithmic growth phase was used for our experiments. The number of colony-forming units (CFU) was routinely determined by plating and incubation at 37˚C for 4 weeks on solid medium.
Human neutrophil isolation
All researches involving human samples were approved by the Ethics Committee of the second Hospital of Tianjin Medical University. Neutrophils were isolated using Ficoll-Dextran method from healthy donors’ blood. [23] Purity was greater than 98% as confirmed by flow cytometry using CD11b (561015) and CD66b antibodies (561927, BD Biosciences). Trypan blue exclusion showed the viability to be > 95% for all preparations. Cell morphology was inspected microscopically to rule out cell preparations that were activated during isolation.
Experimental protocol
The main objective of this study was to investigate the ability of BCG-stimulated neutrophils to produce NETs. Briefly, the experiments were divided into three groups: unstimulated group, BCG-stimulation group, and PMA-stimulation group. The formation of NETs at different time points (0, 30, 180 and 360 min) was observed by optical microscope, Confocal Laser Scanning Microscope (CLSM) and Scanning Electron Microscopy (SEM), and the composition of NETs was analyzed. Then NETs and ROS were quantified by staining extracellular DNA and the cell permeable fluorescent dye respectively. The levels of NETs and ROS were detected after combined BCG stimulated neutrophils with different pathway inhibitors. FITC-labeled BCG was used to observe the capturing by NETs. Finally, two studies were conducted to detect NETs formation in mice. It was observed in mouse urine and subcutaneous tumor after BCG perfusion respectively. To observed NETs in mouse urine, perfusion of BCG into bladder was performed. The formation of NETs in subcutaneous tumor was observed after injection of BCG into tumor.
Stimulation of neutrophils by BCG/PMA
5 × 105-106 cell/mL freshly isolated neutrophils were seeded gently into culture plates on anti-peeling coverslips (S1815-1PAK, Sigma-Aldrich), and allowed to settle in complete RPMI supplemented with 2% FCS (16140071, Gibco BRL) for 2 h, at 37°C, 5% CO2. Neutrophils were respectively stimulated with 50 nM phorbol 12-myristate 13-acetate (PMA) (79346-1MG, Sigma-Aldrich), BCG (MOI=10) or left unstimulated, for indicated time. The total amount of DNA/protein extruded by a given number of cells stimulated by BCG/PMA are used for comparison. As a positive control, the potent inducer of NETs-PMA at this concentration induces typical NETs.
CLSM and SEM visualization of NETs
As film adherent on slips, NETs specimens were gently fixed with 4% paraformaldehyde (P8430, Solarbio), and the percentage of NETs-forming neutrophil was determined by staining DNA with DAPI (D9542-1MG, Sigma). 10 different fields were observed in each triplicate. To evaluate NETs composition, immuno-fluorescence assays were made with respectively antibodies for citrullinated Histone H3 (cit-H3) (ab219407), primary antibodies against NE (ab14188) or isotype IgG (ab27478). Then observed under FV1000 confocal microscope (Olympus, Tokyo, Japan), and calculated using IPP software, as described. [9, 24] Meanwhile, NETs formed in the coculture were observed, after treatment with 50 U/mL DNase I (M6101, Promega) or 50 μg/mL proteinase K (P5568-1ML, Sigma) for 30 min.
The samples were fixed in 2.5% glutaraldehyde and then post-fixed with 1% osmium tetroxide/1% tannic acid. After dehydration with series of ethanol and critical-point drying, the specimens were coated with platinum and analyzed by S2460 N SEM (Hitachi, San Jose, CA).
NETs quantitation
At the indicated time, NETs scaffold in coculture was dismantled by digestion with 250 mU/mL micrococcal nuclease (LS004797, Worthington Biochemical Corp.), which can cleave naked DNA and not act upon histone-attached DNA, ensuring NETs isolation with minimal degradation. The supernatant of stimulated cells was collected and extracellular DNA was then stained with 2.5 μM Sytox Orange (S34861, Molecular Probes) for 10 min at room temperature. The cell-impermeable compound SYTOX orange becomes fluorescent only when interacting directly with DNA. Quantification was done using a fluorometer (Synergy H1 Hybrid Reader, BioTek) every min for up to 300 min. Another way was to calculate percentage of NETotic cells per 100 cells.
Preparation of cell-free NETs and quantification of DNA
The methods of quantification of DNA and protein were descripted previously.[22] Briefly, Neutrophils (1×105/mL) were stimulated with BCG (MOI=10) for 4 h, the medium was removed and cells were gently washed. After addition of 1 mL RPMI to the adherent film and vigorous agitation followed by centrifugation, the supernatant was collected. DNA and protein were quantified using Picogreen dsDNA kit (P11495, Invitrogen), according to the instructions.
Quantification of intracellular ROS
Intracellular ROS production was monitored using the cell permeable fluorescent dye, DCFH-DA (Invitrogen). Neutrophils at 5×106/mL were incubated with 5mM DCFH-DA for 30 min and then harvested. The fluorescence intensity was measured using a reader (488/525 nm, BD Biosciences), every 3 min, for 40 min. At minute 20, neutrophils were infected with non-opsonized BCG at MOI of 10:1, or neutrophils alone as control. Activation with 20 nM PMA was used as an activation control.
Inhibition of ROS, NADPH oxidase and the pathways possibly involved in NETs formation
When indicated, PMNs were pretreated for 30 min and cultured in the presence of 10mM Acetylcysteine (ROS scavenger, Selleck), 20 µM diphenyleneiodonium (DPI, NADPH oxidase inhibitor; Sigma-Aldrich), 10 mM SB600125 (JNK inhibitor, Cayman), 100 nM Wortmannin (PI3K inhibitor, Cayman), 100 nM Sotrastaurin (PKC inhibitor, Selleck), 10 nM U0126 (MEK inhibitor, Cayman), 10 mM SB203580 (p38 inhibitor, Selleck) for the duration of the experiment.
Capturing and killing by NETs
BCG was FITC–labeled as described previously. [25] Neutrophils were seeded and incubated with BCG at MOI of 10:1, for dedicated time. After fixation with 4% paraformaldehyde, DNA fibers were stained with DAPI. CLSM and SEM were performed to observe them. Using the reported protocols, [15] we examined BCG-killing activity by NETs. Neutrophils were pre-incubated with BCG or PMA for 4 h to induce NETs. The medium was carefully replaced with 10 mg/mL cytochalasin D (Cyt-D), the actin inhibitor (Sigma-Aldrich). Then, BCG (MOI=1) was added to incubate for 6 and 24 h, with S.aureus (ATCC25923) as the positive control. After CFU counting, survival was determined as percentage of bacteria with NETs to the ones without NETs.
NETs formation in mouse urine and tumor after BCG treatment
6-7-week-old C57BL/6 mice were from HFK Bioscience Co. Ltd., Beijing, China. All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986. The murine bladder tumor cell line MB49 is a kind gift from M.A. O’Donnell (University of Iowa, Iowa City, IA). The mice receiving subcutaneous inoculation of 106 murine syngeneic MB49 cells were used to evaluate the formation of BCG-induced NETs in vivo. Briefly, for intravesical BCG, the bladder-emptied model mouse was anesthetized and fixed. The BCG suspension was aspirated with a syringe, and slowly inserted into the urethra of the model mouse with a perfusion needle. Single BCG treatment was performed by perfusion of 105 CFU into bladder, or injection of 104 CFU into tumor, then urine and tumors were continuously collected for evaluation of NETs formation. Part of mice were sacrificed to track BCG in bladder wall by auramine ‘O’ staining. Three mice were used once per test.
In freshly collected urine, NETs concentration was determined by fluorescence imaging, after centrifugation and fixation with 2% PFA, staining with DAPI, anti-cit-H3 Ab (ab219407), and anti-NE primary Ab (ab205670). 5 μm tissue sections were used for routine treatment and blockage with 0.2% horse serum, then incubated with primary antibodies against MPO (ab9535) and NE (ab205670, Abcam), followed by respective Alexa Fluor®488 and Alexa Fluor®555 labeled secondary Ab (A-11008, A-21434, Invitrogen). Lastly, DNA was stained using DAPI, for observation under CLSM.
Statistical analysis
Unless otherwise stated, all data are presented as mean ± SD of at least three independent experiments. Where appropriate, either two-tailed Student’s t-test or one-way ANOVA followed by Tukey’s multiple comparison test were used. P < 0.05 was considered statistical significance. SPSS 20.0 software was used for statistical analysis.