Materials and Methods
Mice
Experiments were conducted after obtaining permission from the Israel
Committee for Animal Experiments (IL-32-06-2013, IL-25-5-2016,
IL-39-8-2017). BALB/c and C57BL/6 mice were purchased from Harlan
(Jerusalem, Israel), A1R knock-out (KO) mice
(A1R-/- on C57BL/6 background) and
A2AR knock-out mice
(A2AR-/- on BALB/c background) were
purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Mice were
housed under specific pathogen-free conditions and maintained in the
vivarium of Ben-Gurion University. All experiments were approved by the
Ben-Gurion University Committee for Ethical Care and Use of Animals in
Experiments.
Agonists, Antagonists, and
Inhibitors
A1R agonist
2-chloro-N6-cyclopentyladenosine (CCPA),
A2AR agonist 2-p -(carboxyethyl)
phenethylamino-5’-N-ethylcarboxamideadenosine hydrochloride (CGS21680),
and A1R antagonist 8-cyclopentyl-1,3- dipropylxanthine
(DPCPX) were purchased from Sigma-Aldrich (Rehovot, Israel).
Lupus Model
Pristane, a
natural saturated terpenoid alkane obtained
primarily
from shark liver
oil, was shown to induce a lupus-like disease in mice (14). Injection
of pristane into the peritoneal cavity results in a chronic peritonitis
associated with high tissue levels of interleukin 6 (IL-6) (15), that
leads in a slow process to lupus-like disease (16).
Mice were injected i.p. with 0.5 ml of pristane (Sigma Aldrich) and
followed weekly for external signs of lupus such as alopecia, chronic
wounds, or death. Spleen, blood, and peritoneal lavage were collected at
sacrifice (6h, 24h, 48h, 6 days, 10 days, or 8 months after injection)
(17).
Anti-Double-Strand DNA
(dsDNA)
Disease activity was considered according to anti-dsDNA antibodies (18).
Serum was separated by centrifugation at 4°C at 3000 rpm for 10 min, and
serum anti-dsDNA levels were analyzed using a murine ds-DNA standard
enzyme-linked immunosorbent assay (ELISA) kit (Alpha Diagnostics Inc.;
San Antonio, TX, USA).
Differential blood cell
counts
Blood samples of 200 µl in heparin-coated tubes were counted with an
ADIVA 2120 blood count device (Siemens; Munich, Germany).
Cell-free DNA assay
Peritoneal lavage was performed with 5 mL of PBS at the experiment
endpoint. cfDNA was quantified, as previously described, using our rapid
SYBR® Gold fluorometric assay (19).
Splenocytes production
For mRNA levels, spleens were harvested, and cells were collected and
treated with RBC lysis solution (5 Prime Inc.). Cells were incubated in
a petri dish at 37ºC with medium for 1h. They were then washed, adhered
cells were collected, and RNA was extracted using a PerfectPure RNA
Tissue Kit (5 Prime Inc.).
mRNA Analysis by Quantitative PCR
RNA was extracted using a Perfect Pure RNA Tissue Kit (5 Prime Inc.).
cDNA was prepared using a high capacity cDNA reverse transcription kit
(Applied Biosystems; Foster City, CA, USA).
Quantitative real-time polymerase chain reaction (qPCR) assays were
performed with a Fast SYBR Green Master Mix (Applied Biosystems) on a
StepOne Plus real-time PCR machine (Applied Biosystems) with the
following mouse-specific primers: RPL-12 sense 5‘-ATG ACA TTG
CCA AGG CTA CC-3‘, anti-sense 5‘-CAA GAC CGG TGT CTC ATC TGC -3‘;A1R sense 5‘-TAC ATC TCG GCC TTC CAG GTC G-3‘,
anti-sense 5‘-AAG GAT GGC CAG TGG GAT GAC CAG-3’;A2A sense 5’- CGC AGG TCT TTG TGG AGT TC-3’,
anti-sense 5’-TGG CTT GGT GAC GGG TATG-3’;
Type 1 Diabetes Model
(TID)
We employed the model of low-dose streptozotocin (STZ, Sigma-Aldrich) to
induced TID in all of our experiments. In this model, diabetes develops
only when STZ induces both β-cell toxicity and T-cell-dependent immune
reactions (20). We employed a regimen involving multiple administrations
of low-dose STZ in mice (21). Diabetes was induced in 8-week-old C57BL/6
mice of both sexes by i.p. injection of STZ (50 mg/kg in citrate buffer)
on five consecutive days. Blood glucose levels were measured using a
glucometer (Accu-Chek Aviva, Roche Diagnostics; Indianapolis, IN, USA).
Regularly, in all STZ-injected mice throughout the experiment, animals
with glucose levels >200mg/dl for two consecutive days were
considered to be diabetic (22).
Regulation of NETosis by
Adenosine
Differentiated HL-60
cells
HL-60 cells (CCL240; American Type Culture Collection) were grown in
RPMI 1640 and supplemented with 10% heat-inactivated fetal calf serum
(FCS), 2 mmol/l L-glutamine, 100 U/ml penicillin, and 100 μg/ml
streptomycin (Biological Industries; Bet Haemek, Israel). HL-60 cells
were differentiated into neutrophils by culturing the cells in medium
containing 5µM retinoic acid (RA, Sigma-Aldrich) for 72h.
Differentiation was confirmed by detection of Surface CD11b, which is an
early marker of neutrophil differentiation in HL-60 (23).
Differentiation was confirmed when CD11b expression levels were at least
90%. Untreated HL60 cells stained with the isotype control were used as
background for undifferentiated cells.
Mice neutrophils ex-vivo
Bone marrow (BM) cells from the os femoris and tibia were collected and
treated with red blood cell (RBC) lysis solution (5 Prime Inc.;
Gaithersburg, MD, USA). Neutrophils were then isolated via density
gradient according to Swamydas et al. (24) using centrifugation with
Histopaque 1077 and 1199 (Sigma-Aldrich). Cells were washed twice, and
neutrophils were counted after trypan blue staining using a Neubaur
hemocytometer. Cells were grown in RPMI 1640 and supplemented with 10%
heat-inactivated FCS, 2 mmol/l L-glutamine, 100 U/ml penicillin, and 100
μg/ml streptomycin (Biological Industries; Bet Haemek, Israel).
NETs assay
RA-differentiated HL-60 neutrophils or BM-isolated cells were seeded
(2x105 per well) in 96-well plates. Cells were
pre-incubated with (or without) adenosine agonists (A1R
specific agonist CCPA, 1nM. or A2AR agonist GCS, 30nM)
for 30 min. Then they were treated with 200nM phorbol 12-myristate
13-acetate (PMA, Sigma-Aldrich) and 0.03%
H2O2 for 3h (25, 26). For DNA detection,
Sytox green dye (Molecular Probes, Invitrogen AG; Basel, Switzerland)
was used.
2.8 Statistical Analysis
All comparisons between groups were carried out by a Mann–Whitney
nonparametric t-test or by a one-way ANOVA followed by a Tukey post-test
using Prism 6 software (GraphPad; San Diego, CA, USA). p values
below 0.05 were considered significant. Data are presented as mean ± SD,
unless mentioned otherwise.