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.