Isolation of NK cells and Cell culture
Human NK cells were isolated from PBMCs with Dynabeads® Untouched™ Human NK Cell-Kit according to the manufacturer`s instructions (Thermo Fischer Scientific). For experiments with fresh NK cells, isolated NK cells were rested in IMDM GlutaMAX™ medium (Thermo Fischer Scientific), 10% FCS, 1% penicillin/streptomycin (Thermo Fischer Scientific) overnight and then used for experiments. NK cell isolation purity and viability (7AAD, BioLegend) was confirmed by flow cytometry. NK cells were defined as CD3- and CD56 + cells. To culture cells, isolated NK cells were seeded in 96-well round-bottom plates (Nunc) at a density of 1.5 * 106 /mL with irradiated feeder cells (K562-mbIL15-mbIL21-41BBL) and 200 U/mL IL-2 (NIH Cytokine Repository). On day 8, NK cells were re-stimulated with 200 U/mL IL-2. In the next weeks, NK cells were split to a density of 1.5-2 * 106 /mL, cultured in the presence of 100 U/mL IL-2 and after four weeks used as pre-activated, cultured NK cells.
Cell lines
K562 cells were cultured in IMDM medium (Thermo Fischer Scientific), 10% FCS, 1% penicillin/streptomycin. A549-H2Bj-GFP cells have been kindly provided by Dr. Slava Ziegler and were cultured in DMEM (Thermo Fischer Scientific), 10% FCS, 1% penicillin/streptomycin.
Acute and chronic β2AR stimulation
NK cells were treated with epinephrine (Sigma-Aldrich) or indacaterol (Cayman Chemicals) at a concentration of 1 µM. Acutely stimulated NK cells were treated 0.5 h before or directly in the experiment. For chronic β2AR stimulation, NK cells were treated every 24 h for a total of 96 h with a β2AR agonist.
Flow cytometry
Protein expression levels were evaluated using the BD LSRFortessa flow cytometer. PBMC and NK cell adrenergic receptor expression levels were examined by incubation with alpha-1A AR (PolyAb, Proteintech), alpha-1B (471802, Invitrogen), alpha-1D AR (PolyAb, Invitrogen) or β2AR (6H8, Abcam) at RT for 20 min followed by secondary antibody staining (goat-anti- mouse PE or goat-anti-rabbit PE (Jackson ImmunoResearch)). Cells were stained with the following reagents: 7AAD (Invitrogen), CD16-PE (3G8, Biolegend), GLUT-1 (202915, R&D systems), GLUT2-AF647 (199017, R&D systems), GLUT3-AF700 (202017, R&D systems). Data were analyzed using FlowJo (version 10) software.
IFNγ ELISA, degranulation assay
Nunc MaxiSorp™ plates were prepared by antibody immobilization. Indicated antibodies (NKG2D (149810; R&D Systems), 2B4 (C1.7; Beckman Coulter), NKp30 (produced in our lab), CD16 (3G8, BioLegend)) were incubated overnight at 4°C at a concentration of 1 µg/mL. Next day, 0.2 * 106 NK cells (fresh or cultured) were pretreated with epinephrine or indacaterol (1µM) for 0.5 h and subsequently stimulated with plate-bound antibodies or IL-12/-18 (0.5 ng/mL, R&D / 2.5 ng/mL, MBL Life Science) for 5 h at 37°C, 5%CO2. The supernatant was collected after centrifugation (5 min, 500×g) and stored at -20°C. The secreted IFNγ concentration was determined by human ELISA MAX™ Deluxe Set Human IFN-γ from BioLegend according to manufacturer’s instructions. For degranulation assay, NK cells were pre-treated with β2AR agonists and activated by plate-bound antibodies. During 3 h incubation, NK cells were stained with anti-CD107a-PE-Cy5 (H4A3, Biolegend) and analyzed by flow cytometry.
IncuCyte S3 microscopy based killing assay
IncuCyte S3 live-cell analysis was conducted at 37°C and 5% CO2 using 10x magnification. A549-H2Bj-GFP cells were seeded in a density of 3.5 * 10^3 cells/well and incubated in RPMI1640 media overnight. On the second day, pretreated NK cells were added in an E:T ratio of 1.5:1. NK cells were either acutely or chronically treated with indacaterol (1µM) or DMSO (Thermo Fischer Scientific) as solvent control. After 8 h of co-incubation, the plates were washed with PBS and quantitatively analyzed for GFP fluorescence by IncuCyte 2019B Rev Software. A549-H2Bj-GFP without NK cells were used as control and for calculation of the specific lysis.
$$\% specific lysis=100* \frac{\left(A549+NK\right)-\left(A549 control\right)}{0-\left(A549 control\right)}$$
Serial killing
Microscope based serial killing analysis has been performed as described 41. Briefly, K562 cells were seeded on a microchip with SYTOX™ blue dead cell stain (1µM, Thermo Fischer Scientific) in IMDM medium. NK cells were stained with CellTracker Red (5µM, Thermo Fischer Scientific), pretreated with DMSO or indacaterol (1µM) and subsequently added to the microchip. Time-lapse live-cell microscopy was immediately started using a Zeiss Axio Observer Z1 7 microscope equipped with a 20×/0.8 Plan-Apochromat objective and an incubation chamber with environmental control (37°C, 5% CO2, and humidity device S1). Images were acquired every 3 min for 16 h.
Ligand complex adhesion assay (LC-AA)
The assay was performed essentially as described 42. Briefly, ICAM-1–Fc complexes were prepared by mixing 50 ng/mL recombinant human ICAM-1–Fc chimera (R&D Systems) and F(ab)2 fragments of goat anti-human Fcγ fragment specific antibody (80 ng/ml, PE labeled; Jackson ImmunoResearch) in buffer without cations for ≥ 20 min at room temperature. NK cells were incubated with 1 µg/ml anti-NKG2D (149810; R&D Systems) and anti-2B4 (C1.7; Beckman Coulter). After washing, cells were resuspended in buffer containing indacaterol (1µM), DMSO or epinephrine (1µM) and ICAM-1–Fc complexes were added (dilution 1:20). A goat anti-mouse IgG (Dianova) was added to a final concentration of 2.3 ng/ml for Ab cross-linking. Cells were fixed and analyzed by flow cytometry. When indicated, the cells were additionally treated with inhibitors (10 µM H89 (Cayman Chemicals) or 25 µM ESI09 (TOCRIS)). In re-stimulation experiments, inhibitors (10 µM H89 or Barbadin 50µM (MedChem Express)) were kept in media for 24 h and were washed away before LC-AA and second β2AR stimulation.
xCELLigence based detachment assay
E-plates were coated with 7 ng/mL goat anti-mouse antibody (Dianova) followed by 2 ng/mL of recombinant human ICAM-1–Fc chimera (R&D Systems). After addition of 100,000 NK cells per well, impedance was measured for 2h in 10 min intervals. When the cell index reached a plateau, epinephrine, indacaterol (1 µM) or medium, DMSO were added and the cell index was determined every 2 min. For the analysis, the cell index was normalized to the point of β2AR stimulation. Experiments were performed on xCELLigence® RTCA DP.
cAMP ELISA
Cultured NK cells were chronically stimulated with indacaterol (1 µM) or DMSO for 96 h. When indicated, NK cells were pretreated with Pertussis toxin (100ng/mL, (TOCRIS)) for 30 min at 37°C before β2AR stimulation. After 95 h, NK cells were incubated for 0.5 h in 100µM IBMX (Cayman Chemicals) to inhibit phosphodiesterases before last indacaterol incubation step (0.5 h). 1x106 NK cells were lysed in 100 µL 0.1 M HCL, and the supernatant was stored at -20°C until cAMP analysis. The ELISA was performed according to manufacturer Cayman Chemicals instructions.
Immunoprecipitation and Western blot
For 2B4 phosphorylation analysis 4x107 NK cells were incubated for 20 min at room temperature with 2B4/NKG2D antibodies (10µg/mL). After a washing step, NK cells were treated with PBS or epinephrine ± propranolol (2µM each) and activated by crosslinking the antibodies by a goat anti-mouse antibody (10µg/ml, (Dianova)) for 0 min or 5 min at 37°C. The reaction was stopped by the addition of ice-cold PBS. Cells were washed, lysed and the supernatant was transferred to pan mouse IgG beads (approx. 7x106) and rotated for 1h at 4°C. Beads were washed in lysis buffer and dried. Dried beads were shortly boiled for 5 min at 95°C in RSB buffer and solution was used for SDS PAGE. Western blotting was essentially performed as described 43.
Metabolic extracellular flux assays
The Seahorse XFe96 Analyzer and Wave software was used to measure the NK cell glycolysis (extracellular acidification rate-ECAR) and oxidative phosphorylation (oxygen consumption rate-OCR) via extracellular flux. Cultured NK cells (250,000 cells/well) were immobilized for 30 min at 37°C without supplemented CO2 in Seahorse RPMI media (10mM glucose, 1mM pyruvate, 2mM glutamine, Agilent Technologies) on Poly-(L)-Lysine (Sigma-Aldrich) coated cell culture plates. The measurement steps were set to a 3 min mixing/ 3 min measurement interval. Epinephrine (1µM), indacaterol (1µM), DMSO or propranolol (1µM) were injected as indicated before NK cells were activated by CD16 antibody (1 µg/mL, 3G8) or cytokines IL12/15/18 (50 ng/mL (R&D Systems) /250 ng/mL (Pan Biotech) /250 ng/mL MBL Life Science). After 160 min, the measurements were terminated and normalized to the timepoint of β2AR agonist addition.
Transcriptomics
Cultured NK cells from three different donors were chronically stimulated for 96 h with epinephrine (1µM), indacaterol (1µM) or DMSO as described above. NK cells treated with medium were used as reference. The transcriptomic analysis was carried out by Eurofins GATC Biotech GmbH. The expression analysis of the RNA-Seq reads were aligned to the reference transcriptome using Bowtie alignments. The variant analysis was done by GATK Haplotype Caller. The aligned reads were used by MATS to detect alternative splicing events. The R package GenomicFeatures 44 was used to summarize transcript reads at gene level. For pre-filtering, genes with a mean count of less than 1 read across the nine samples (6,893) were removed, leaving 16,566 genes for further analysis. Differential gene expression analysis was performed using the R package DESeq2 45. A general linear model including the variables replicate and treatment was fitted to determine differentially expressed genes (DEGs). DEGs were then calculated for the comparisons "epinephrine vs. untreated" and "indacaterol vs. untreated", respectively. For more reliable effect estimates, adaptive shrinkage was applied 46. This leads to shrinkage of log2-transformed fold-changes (log2FCs) towards zero if expression changes are mostly due to noise, whereas relevant log2FCs are preserved. For each comparison, a gene was considered to be differentially expressed if the effect size satisfies log2FC > 1 for upregulation (log2FC < -1 for downregulation) and the estimate is significantly different from zero (i.e., no effect) with a false discovery rate (FDR)-adjusted p value padj < 0.05.
Proteomics -LC-MS/MS
Cultured NK cells (106 cells per sample) were chronically stimulated for 96 h with epinephrine (1µM) or indacaterol (1µM). NK cells only treated with medium were used as reference. After chronic stimulation, supernatant was discarded and cells were frozen at -80°C in 10%DMSO/FCS. Cryocultures were resuspended in 80% methanol, lysed at 4°C by ultrasonication (Bioruptor, Diagenode) and centrifuged (21000×g, 5 min, 4°C). Next, pellets were proteolytic digested by Trypsin and prepared using Gel-aided sample preparation 47. The peptides were separated by LC (Ultimate3000 RSLC, Thermo) according to hydrophobicity in a reversed-phase column (Acclaim PepMap, 300µM ID – 15cm) and pre-column trapping. The MS/MS analysis were done in two steps as described 48. Briefly, in the first step a MS library is generated with pooled samples using the TOP30-method. In the second step, samples were analyzed by SWATH method. The data analyses were done using PeakView and MarkerView (both Sciex).
Asthma patients and healthy subjects
The asthma patients (n = 10) and healthy control cohort (n = 7) were recruited from the department of Pulmonary Medicine, University Medical Center Essen-Ruhrlandklinik, Essen, Germany. Three additional healthy control subjects were recruited at the Leibniz Research Centre for Working Environment and Human Factors (IfADo), Dortmund, Germany. The blood sampling from asthma patients was approved by the Westdeutsche Biobank Essen (University Medical Center Essen, approval number 22-WBE-142) and the sampling from healthy volunteers by the ethics committee of the Leibniz Research Centre for Working Environment and Human Factors (IfADo), Dortmund. Written informed consents for participation and publication were obtained. The blood samples were collected in heparin vials and analyzed the same day. PBMCs were isolated and functionally checked by degranulation assay, IFNγ ELISA and LC-AA. Additionally, the PBMC composition were determined by Cytek Aurora spectral flow cytometry using the following antibodies: CD3-BUV563 (UCHT1, BD Biosciences), CD56-BUV 805 (B159, BD Biosciences), CD226-AF700 (DX11, BD Biosciences), CD314-AF700 (FAB139N, R&D systems), CD335-BV421 (9E2), CD244-FITC (C1.7), CD336-PerCP-Cy5.5 (p44-8), CD16-PE Dazzle (3G8), CD337-APC-Fire750 (p30-15), Zombie NIR (all Biolegend). PBMCs were incubated with live/dead marker Zombie NIR for 15 min, washed and subsequently stained with antibodies for 20 min at 4°C.
Statistics
Statistical analysis was performed using GraphPad Prism version 9.