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ELECTRONIC SUPPLEMENTARY MATERIAL
METHODS
Study population. The study was approved by the local ethical committee and carried out in
accordance with the principles of the Declaration of Helsinki as revised in 2008. All subjects
provided written informed consent. Type 2 diabetic (T2D) patients were recruited from the
outpatient diabetes clinic of the University Hospital of Padova. Non diabetic subjects were selected
among individuals presenting at the same outpatient clinic for a cardiometabolic screening.
Subjects were enrolled provided they were free from any acute disease or infection and did not
report active inflammatory conditions (e.g. rheumatic diseases), recent trauma or surgery,
pregnancy/lactation. The following data were collected for all participants: age, sex, body mass
index, history of hypertension, smoking habit, prevalence of cardiovascular disease, and
medications. We also collected a fasting blood sample for determination of HbA1c and lipid profile,
and a spot urine sample for determination of albumin/creatinine ratio (ACR). Coronary artery
disease (CAD) was defined as a past history of myocardial infarction or angina, or angiographic
evidence of >50% obstruction of epicardial coronary arteries, or a positive myocardial perfusion
stress test. Peripheral arterial disease (PAD) was defined in the presence of claudication or rest
pain, or evidence of >50% obstruction in lower extremity arteries or an ankle-brachial index of less
than 0.9. Cerebrovascular disease (CerVD) was defined as a past history of stroke or transient
ischemic attack, or evidence of >30% carotid artery stenosis, or carotid endarterectomy. Prevalent
atherosclerotic cardiovascular disease (CVD) was defined as either CAD, or PAD or CerVD
isolated or in combination. In diabetic patients, we also collected disease duration, prevalence of
retinopathy (defined by digital funduscopic examination), neuropathy (defined by typical signs and
symptoms, eventually confirmed by vibration perceptive threshold and/or electromyography), and
nephropathy (defined as an albumin excretion rate >30 mg/g creatinine and/or estimated
glomerular filtration rate <60 ml/min/1.73 m2).
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Circulating mono- and oligonucleosomes. The Cell Death Detection ELISAPLUS (Roche
Diagnostics, IN, USA) was used. All reagents were prepared at room temperature, and the serum
samples, positive controls and background control added into the microplate, covered with
adhesive film, and incubated for 1 hour at room temperature with the immunoreagent containing
anti-histone-biotin, anti-DNA-POD and incubation buffer. After washing, the reaction was run at
room temperature for 20 min avoiding direct exposure to intense light, stopped by adding stop
solution, and absorbance at 490 nm wave length read on a spectrophotometer (BIO-RAD Model
680 microplate reader, manager software version 5.2.1).
Circulating cell-free double strand DNA. The serum was diluted with an equal volume of saline
and an equal volume of phenol was added to the DNA solution to be purified in a 1.5 ml
microcentrifuge tube. Tube contents were mixed vigorously until formation of an emulsion. Tubes
were centrifuged at 80% maximum speed for 1 min at room temperature. At the interface, a light
coloured protein layer was visible. The top (aqueous) phase containing the DNA was carefully
removed and transferred to a new tube. An equal volume of chloroform was added and the mixture
shaken vigorously and centrifuged at max speed for 5 min at room temperature. Then, 1/10 volume
of 3 M sodium acetate (pH 5.2) was added to the solution of DNA and mixed by vortexing. 2.5 vol
of ice-cold 100% ethanol was added and mixed by flicking the tube several times, placed in dry ice
for 5 min, spun for 25 min in a fixed-angle microcentrifuge at high speed at 4°C, and the
supernatant removed. 1 ml of room temperature 70% ethanol was then added, the tubes inverted
several times, and centrifuged at high speed at 4°C, the supernatant removed and air dried for 15
min. The DNA pellet was resuspended in 100 μl of ultra pure water. The quantification of dsDNA
was carried out using the Qubit® 2.0 Fluorometer (Life Technologies) according to the instruction
manual of the instrument.
Serum neutrophil elastase. The polymorphonuclear Elastase Human ELISA Kit (Abcam) was
used. All reagents were prepared at room temperature, and serum samples prediluted 1:100. The
microplate was washed and prepared according to the manufacturer’s instructions. Standards,
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samples, high and low controls, HRP and the TMB substrate solution were then added to the wells.
The reaction was conducted for 20 min at room temperature in the dark, stopped by adding stop
solution and absorbance at 450 nm wave length read on a spectrophotometer (BIO-RAD Model
680 microplate reader, manager software version 5.2.1).
ELISA. Serum concentrations of IL-6 and TNF-α were determined using the Human IL-6 Platinum
ELISA (Affymetrix eBioscience, cod. BMS213/2) and the Human TNF-alpha Platinum ELISA
(Affymetrix eBioscience, cod.BMS223/4), respectively, according to manufacturer’s instructions.
In vitro analysis of NETosis and release of NETs. White blood cells were freshly isolated from
blood of n=10 healthy donors. After red blood cell lysis, cells were seeded on 10 mm glass
coverslips on 24-well plates in 500 μl of RPMI no glucose, supplemented with 10% serum,
glutamine and penicillin-streptomycin antibiotic at a density of 105 cells per well. Plates were
incubated for 2 hours at 37°C to allow adhesion of cells with different stimuli. We used phorbol 12myristate 13-acetate (PMA) 50 nM, and/or 20% dimethyl sulfoxide (DMSO) as positive controls. In
separate experiments, glucose was supplemented to a final concentration of 5 and 25 mM.
Mannitolo 25 mM was used as osmotic control. After 2 hours of incubation, the cells were fixed
with 4% PFA.
For immunofluorescence, coverslips with fixed cells were removed from the plates issued on drops
kept on parafilm. After washing with PBS, the cells were permeabilized for 1 min with 0.5% Triton
X100 in PBS, washed in PBS and blocked for 20 min with blocking buffer, containing 3% goat
serum, 1% BSA, and 0.05% Tween 20 in PBS. Cells were then stained with a mouse anti-human
PL2-3 mAb, directed against the subnucleosomal complex of Histone 2A, 2B and chromatin (kind
gift of Volker Brinkmann, Max Planck Institute for Infection Biology, Berlin, Germany), with a
polyclonal rabbit anti-human neutrophil elastase (Calbiochem 481001) in blocking buffer for 1h at
37°C. In parallel experiments, cells were stained with a propidium iodine, or a monoclonal rabbit
anti-human histone H2B (clone EP957Y, Millipore) or a polyclonal rabbit anti-human citrullinated
(R2+R8+R17) histone H3 to visualize chromatin. After washing with PBS the secondary antibodies
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were applied for 30 min (Cy3 goat anti-mouse, AF488 goat anti-rabbit, Jackson Laboraratory).
Cells were then washed with PBS and stained with Hoechst 33342 (100 ng/ml, Sigma-Aldrich),
washed with PBS and mounted with Elvanol. Neutrophils were identified by typical nuclear
morphology and staining for elastase. As >90% of polymorphonuclear cells in peripheral blood are
neutrophils, the analysis carried out using double IF images without elastase staining were not
biased to a significant extent by the presence of eosinophils and basophils. It has been previously
shown that anti-chromatin antibodies bind more readily to decondensed chromatin present in the
nuclei of cells undergoing NETosis [1]. Thus, netting cells were semi-automatically identified by
relating the fluorescence signals of the anti-chromatin antibodies to the signal of Hoechst 33342.
To this end, a customized procedure was run using using ImageJ/Fiji software (1.48r, NIH, USA): a
line of interest (LOI) was drawn with care to cross the cell borders intersecting 2 lobes of the
nucleus. Relative intensity signal was plotted for each channel separately (Hoechst 33342, blue;
Chromatin, red; Elastase, green) against distance in pixels along the LOI. Cells undergoing
NETosis were identified by a chromatin signal increase and spread out of the nuclear region. In
separate proof-of-concept analyses, a 2-dimension region of interest (ROI) was used and a 3D
histogram was constructed with relative colour intensity, representing fluorescence. For the
analysis of netting cells in replicate experiments, a semi-automated high-throughput analysis was
conducted as previously described using the colour threshold and particle measure functions of
ImageJ/Fiji to identify and record ROIs of 0.1-1.0 circularity score and 250-5200 pixel area (equal
to 8-36 μm2). ROI areas in the cyan-blue and the red channels with thresholded brightness were
recorded and transferred to a spreadsheet. The ratio between Hoechst 33342 and chromatin areas
was calculated for each ROI and a value ≥1.5 was used to define netting cells. This ratio was
chosen based on a preliminary comparison with blind manual counting.
After an initial instructive workup with triple staining, it emerged that classical elongated
extracellular filaments inter-connecting neutrophils, and possibly other white blood cells, were
always stained for Hoechst 33342, chromatin and elastase. Therefore, for quantification of NETs in
replicate experiments, the red and green channels were dropped to reduce noise, increase
efficiency and throughput, as NETs are easily visualized by Hoechst 33342 staining alone. Triple-
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stained images were generated and used solely as control images to visually assess and exclude
the influence of non-neutrophil cells or imaging artefacts. NETs were quantified in 10 randomly
selected 40x fields for each experiment and related to the number of neutrophils in each field. In
vitro experiments were replicated 3-5 times. Images were acquired with a Leica DM5000B
microscope, equipped with a DFC300 FX CCD camera.
SUPPLEMENTARY RESULTS
In vitro features of NETosis. We applied the quantitative and semi-automated method proposed
by Brinkmann et al., which overcomes inconsistencies in the methods to assess NETosis in vitro
[1]. Cells were stained with the fluorescent cell permeable nuclear dye Hoechst 33342 (blue),
which binds externally to DNA providing a signal proportional to DNA concentration. Cells were
also stained with the PL2-3 anti-chromatin antibody (red), which recognizes histone 2A and 2B and
binds preferentially to decondensed chromatin. Counterstaining for neutrophil elastase (green),
together with the typical polymorphonuclear shape, allowed distinction of neutrophils from other
blood cells (Figure S1a). After stimulation with PMA for 2 hours, some cells had undergone
NETosis, while some were not (yet) netting. Merged images and morphometric analysis show that
netting cells undergo nuclear remodeling with progressive loose of typical lobation, chromatin
decondensation (evidenced by increased red signal of the PL2-3 antibody), entering of elastase in
the nucleus (evidenced by anti-elastase green signal overlapping with the nuclear area), and
ultimate extrusion of chromatin from the nucleus, indicated by the red signal exceeding and
spreading over the Hoechst 33342 fluorescence. At the same time, filaments of NETs are visible
and stain triple positive for DNA binding dyes and elastase. This late phase of NETosis can also be
visualized by analyzing a 2-dimensional ROI and constructing 3D histograms showing relative
location and intensity of the blue Hoechst 33342 and of the red chromatin fluorescence signal
(Figure S1b). Citrullination of histones is a critical step during NETosis [2], and our morphometric
analysis shows that the signal of the anti-citrullinated histone H3 increases and spreads out the
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Hoechst 33342 signal in netting cells (Figure S1c). These in vitro proof-of-concept experiments
show how neutrophils release chromatin, histones and elastase during PMA-stimulated NETosis.
REFERENCES
1.
Brinkmann V, Goosmann C, Kuhn LI and Zychlinsky A (2012) Automatic quantification of in
vitro NET formation. Front Immunol 3:413
2.
Leshner M, Wang S, Lewis C et al (2012) PAD4 mediated histone hypercitrullination
induces heterochromatin decondensation and chromatin unfolding to form neutrophil extracellular
trap-like structures. Front Immunol 3:307
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SUPPLEMENTAL FIGURE LEGEND
FIGURE S1. Methods for in vitro quantification of NETosis. a) The upper lane shows single
channels and merged images of labeling with Hoechst 33342, anti-chromatin and anti-elastase
antibodies to reveal the typical features of NETotic cells. Yellow rectangles indicate areas
magnified below and showing non-netting, early netting and advanced netting cells. Lines of
interest (LOI) were used to create histograms of each channel. Non-netting neutrophils with the
typical notched nuclear profile show a higher Hoechst 33342 intensity compared to fluorescence
signal produced by the anti-chromatin and anti-elastase antibodies. Cells in the early phases of
NETosis show more irregular nuclear profiles and rising fluorescence intensity of the antichromatin antibody and anti-elastase. In advanced NETosis, the nucleus looses lobulation and the
chromatin signal exceeds and spreads over the Hoechst 33342 stained region. In addition, triple
labeled NET filaments are visible (arrows). b) The LOI can be replaced by 2D regions of interest
(ROI) to construct 3D histograms based on the fluorescence intensity of the Hoechst 33342 and
chromatin signal. c) Analysis of the fluorescence intensity of Hoechst 33342 and anti-citrullinated
histone H3. Two non-netting cells and one cell in advanced NETosis are shown. White scale bar
always set at 20 μm.
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FIGURE S1
a
c
Chromatin +
H2A/H2B
Hoechst 33342
Elastase
Hoechst 33342
Citrullinated H3
Merged
Merged
Early
10
20
30
40
50
60
b
0
10
20
Distance (px)
100
50
0
b
Hoechst 33342
20
40
Distance (px)
50
60
Chromatin +
H2A/H2B
80
250
200
150
100
50
0
NETs
Merged
a) Netting
neutrophil
80
b) Non netting
neutrophil
70
60
50
40
30
20
10
0
0
20
40
Distance (px)
60
c
60
Rel. Fluorescence
150
Rel. Fluorescence
Advanced
200
0
40
Distance (px)
250
Rel. Fluorescence
Advanced
30
Rel. Fluorescence
0
100
90
80
70
60
50
40
30
20
10
0
80
0
10
20
30
40
Distance (px)
50
60
c) Non netting
neutrophil
90
80
70
60
50
40
30
20
10
0
Rel. Fluorescence
90
80
70
60
50
40
30
20
10
0
Rel. Fluorescence
Rel. Fluorescence
a
Negative
0
10
20
30
40
Distance (px)
50
60
90
80
70
60
50
40
30
20
10
0
0
10
20
30
40
Distance (px)
50
60