Ethics Statement
The studies involving human participants were reviewed and approved by the Columbia University Institutional Review Board (IRB) (Protocol # AAAS0172) and conducted under the Declaration of Helsinki principles. Blood collection was conducted with the informed consent of each participant or surrogates.
Isolation of primary human neutrophils
Venous blood was collected from healthy volunteers and critically-ill patients with sepsis into BD Vacutainer blood collection tubes coated with K2EDTA (BD Biosciences, San Jose, CA, USA). Sepsis was defined as an increase in Sequential Organ Failure Asssessment score by 2 or more points [1]. Samples were kept on ice during handling and processed immediately. MACSxpress Whole Blood Neutrophil Isolation Kit (Miltenyi Biotec, Waltham, MA USA) was used for the isolation of neutrophil granulocytes according to the manufacturer’s protocol. Briefly, blood samples were diluted with PBS to the desired volume and incubated with magnetic beads for 5 mins at room temperature (RT) using a tube rotator to label non-target cells. Subsequently erythrocytes were aggregated and sedimented, while non-target cells were removed by immunomagnetic depletion. Residual erythrocytes were lysed for an additional 2 mins using Hybri-Max Red Blood Cell Lysing Buffer (Millipore-Sigma, St Louis, MO, USA), and neutrophils were washed with PBS and pelleted at 1500 rpm.
Neutrophil aging
Freshly isolated human neutrophils were plated into 12-well plates at 500,000 cells/well. Cells were treated with vehicle, 1-10 μM CGS 21680, 10 μM ZM 241385 or 50 ng/ml IFNγ and incubated in DMEM for 24-48 hrs at 37 ºC in a humidified 5% CO2 incubator to mimic aging. 0-minute samples were collected and stained to establish a baseline for flow cytometry.
Flow cytometry
For staining of cell surface markers, cells were labelled with various antibodies listed in Table 2. Stainings were carried out in FACS buffer (1% BSA-PBS with 2mM EDTA) at 4ºC for 30 minutes. Subsequently cells were washed with FACS buffer and labelled with LIVE/DEAD Fixable Dead Cell Stain for 10 minutes at RT. After another washing step the cells were fixed with 4% paraformaldehyde for 20 minutes at RT, washed one time and resuspended in FACS buffer before analysis. All samples were analyzed using BD FACS Canto II flow cytometer (BD Biosciences, San Jose, CA, USA) equipped with 405, 488 and 633nm lasers. At least 50,000 events per sample were recorded using BD FACSDiva™ Software 9.0. Data from the flow cytometer were analyzed using FlowJo version 10.8.1 (FlowJo LLC, Ashland, OR, USA). Results are presented as median fluorescence intensity (MFI) of the gated live single cell population.
Mice
Experimentation on mice was approved by the Columbia University Institutional Animal Care and Use Committee (IACUC)(Protocol #AC-AABL5575) and was conducted in accordance with international guidelines. Male C57BL/6 mice (10-12 weeks old) were purchased from Charles River Laboratories (Wilmington, MA). Animals were housed up to five mice per cage in rooms with a 12h light–dark cycle under nonspecific pathogen–free conditions. A2AARknockout (KO) mice (C57BL/6J background) and wild type (WT) controls were bred and maintained in a specific pathogen-free Charles River animal facility (Wilmington, MA, USA). All mice were housed for at least 1 week before experimental use at the animal facility at Columbia University and had access to regular chow and water ad libitum. Adult age-matched male mice were used for all experiments.
Cecal ligation and puncture
Polymicrobial sepsis was induced by subjecting mice to cecal ligation and puncture (CLP) [34-39]. A2AAR−/− mice or C57BL/6 mice, injected intraperitoneally with either 15 mg/kg ZM 241385 or vehicle 1 h preoperatively, were used for the surgeries. Mice were anesthetized with isoflurane (2%, 1 L/min) and the depth of anesthesia was checked by hind toe pinch reflex. Under aseptic conditions, a 2-cm midline laparotomy was performed to allow exposure of the cecum. Approximately two-thirds of the cecum was ligated with a 3-0 silk suture, and the ligated part of the cecum was perforated twice (through and through) with a 21-gauge needle (BD Biosciences, San Jose, CA, USA). The ligated cecum was gently squeezed to extrude a small amount of feces through the perforation site and was then returned to the peritoneal cavity, and the laparotomy was closed. After the operation, all mice were resuscitated with physiologic saline (1 ml injected subcutaneously) and returned to their cages, where they were provided free access to food and water. The mice were re-anesthetized with isoflurane 16 hrs after the CLP procedure, and blood was collected via cardiac puncture into Eppendorf tubes containing EDTA.
Isolation of primary mouse neutrophils
Blood was collected from mice 16 hrs post-CLP surgery via cardiac puncture. Samples were kept on ice during handling and processed immediately. Erythrocytes were lysed for 5 mins using Hybri-Max Red Blood Cell Lysing Buffer at RT using a tube rotator. Lysis was stopped by dilution with PBS and cells were pelleted at 1500 rpm. Mouse Neutrophil Isolation Kit (Miltenyi Biotec) was used for the isolation of neutrophil granulocytes following the manufacturer’s recommendations. Briefly, cells were resuspended in MACS buffer (0.5% BSA and 2 mM EDTA in PBS) and passed through a 40-µm nylon mesh to achieve a single-cell suspension. Cells were labelled with Neutrophil Biotin-Antibody Cocktail for 10 mins at 4ºC and were then washed and incubated with Anti-Biotin MicroBeads for 15 mins at 4ºC. After another washing step the cells were resuspended in MACS buffer and loaded onto MS Columns placed in a magnetic field. Flow-through, containing unlabeled cells representing the enriched neutrophils was collected. Neutrophils were washed with PBS and pelleted at 1500 rpm.
Neutrophil polarization
A protocol by Ohms et al. [27] was adopted with slight modifications. Components of N1 and N2 polarization cocktails are listed in Table 3. 5 μM Pan Caspase OPH Inhibitor (Millipore-Sigma) was used to prevent naturally occurring neutrophil aging and cell death. Adenosine, an original component of the N2 cocktail was replaced by the more stable and highly specific A2AAR agonist CGS 21680. Hypoxic conditions to achieve an N2 phenotype were mimicked by treatment with 10 μM Daprodustat (Medchem), a potent inhibitor of HIF-prolyl hydroxylase domain enzymes [40]. Freshly isolated human neutrophils were plated into 12-well plates at 500,000 cells/well in DMEM. Cells were treated with N1 or N2 polarization cocktails for 24-48 hrs at 37 ºC in a humidified 5% CO2 incubator. Treatment with 5 μM Pan Caspase OPH Inhibitor and vehicle (DMSO) was used as control and denoted as N0.
RNA isolation and Real-time PCR
Freshly isolated neutrophils were immediately placed in ice cold TRIzol® reagent (Invitrogen, Carlsbad, CA) and stored at -80°C until RNA isolation. Total RNA was extracted according to the manufacturer’s directions, and then quantified by Nanodrop (Thermo Scientific, Wilmington, DE) and assessed for quality by Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Palo Alto, CA, USA). RNA was then reverse transcribed to cDNA with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). Real-time PCR was performed using primers specific for A2AAR, CXCR4, CD95, CD182 or 18S listed in Table 1. Results were normalized to 18S mRNA content.
Statistical analysis
Values in the figures and text are expressed as mean ± standard error of means (SEM) of n observations. Statistical analysis comparing 2 groups was performed by 2-tailed unpaired student t test. When comparing 3 or more groups, one-way analysis of variance (ANOVA) was used, followed by Šídák's multiple comparisons test. GraphPad Prism 9.1.2 was used for data illustration. Results were considered statistically different when p was ≤ 0.05.
Data availability statement
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.