Fifteen atopic asthmatic volunteers (eight male/seven female; aged 18–65 years, average 34.5 ± 10.6 years; BMI 19–32 kg/m2, no oral steroids for > four weeks) were enrolled into the study. Subjects differed between eosinophil (n = 8) and ILC2 (n = 7) experiments and also between activation and inhibition experiments, respectively. Whole blood (200–500 ml) was collected in 3.8% trisodium citrate and was processed within one hour after blood withdrawal. Eosinophil levels in peripheral blood had to be > 0.15 × 106/ml for the eosinophil shape change.
PGD2, 13,14-dihydro-15-keto-PGD2, PGJ2, Δ12-PGJ2, Δ12-PGD2, 15-deoxy-Δ12,14-PGJ2, 15-deoxy-Δ12,14-PGD2, 9α,11β-PGF2 were purchased from Cayman Chemicals (Biomol GmBH, Hamburg, Germany). Fevipiprant (GST0000013789) was provided by Novartis Pharma AG (Basel, Switzerland). Reagents were dissolved in sterile-filtered Hybri-Max dimethylsulfoxide (DMSO, Sigma-Aldrich, Taufkirchen, Germany).
Blood was stored on ice until processing. Granulocytes were isolated as described [18, 37]. In brief, 200 ml blood was diluted 1:3 in Dulbecco’s Phosphate Buffered Saline (DPBS). The blood suspension was incubated with 4% (w/v) dextran-T500 (dilution 5:1, VWR, Hannover, Germany) for 30 min on ice. The upper phase was layered on Ficoll-Paque® (Sigma-Aldrich, Taufkirchen, Germany) and centrifuged (25 min, 300×g, 18 °C). Granulocyte pellets each were resuspended in 500 µl DPBS and erythrocytes were lysed for 40 s with 20 ml ice cold, sterile, endotoxin-free distilled water. The reaction was stopped with 20 ml DPBS (2 × concentrated). After centrifugation (10 min, 300×g, 18 °C), cell pellets were washed with 50 ml DPBS. Granulocytes were resuspended in assay buffer (0.1% bovine serum albumin (BSA) in DPBS, Sigma-Aldrich, Taufkirchen, Germany) to a cell density of 6.25 × 106/ml.
Eosinophil shape change
Granulocytes (80 µl) and assay buffer (10 µl) were incubated in a water bath (5 min, 37 °C). Metabolite solutions (10 µl, final concentration (conc.) 0.01 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 100 nM, 1 µM, 1% DMSO in assay buffer) were added (5 min, 37 °C). The incubation was stopped with 250 µl of 0.25% BD Cell-Fix™ Solution (1:10 with sterile water followed by 1:4 with assay buffer, BD Biosciences, Heidelberg, Germany) and immediate placement on ice (≥ 5 min). Flow cytometric analysis was performed with a Navios 3/10 (Beckman Coulter). Granulocytes were determined by FSC and SSC properties. Eosinophils were discriminated from neutrophils by autofluorescence properties at 560 nm (FL2). 1000 eosinophils were acquired. Eosinophil shape change was calculated as percentage increase of the mean FSC units.
For DP2 inhibition experiments, granulocytes (80 µl) and fevipiprant (10 µl, final conc. 0.01 nM, 0.05 nM, 0.1 nM, 1 nM, 5 nM, 10 nM, 100 nM, 500 nM, 1 µM, 10 µM) were incubated in a water bath (5 min, 37 °C). Metabolite solution (10 µl, EC70 concentration) was added for 5 min (37 °C). The reaction was stopped and cells were analyzed as described above.
Type 2 innate lymphoid cell isolation
Blood was stored at RT until processing. Whole blood (500 ml) was diluted 1:2 in DPBS. PBMCs were isolated using Ficoll-Paque® and SepMate-50 PBMC Isolation tubes (STEMCELL Technologies, Grenoble, France) following manufacturer’s protocol. PBMCs were pooled, washed with 50 ml DPBS and centrifuged (5 min, 300×g, 4 °C). T cells, B cells and monocytes depletion enriched ILC2s using CD3, CD14 and CD19 MACS separation beads (Miltenyi Biotech, Bergisch Gladbach, Germany) and LD columns following manufacturer’s protocol. Enriched cells were centrifuged (5 min, 300×g, 4 °C) and resuspended in staining buffer containing 5% fetal bovine serum (FBS) and 2 mM ethylenediaminetetraacetic acid (EDTA) in DPBS. Cells were stained for 15 min at RT with a PerCP-Cy5.5-labeled lineage cocktail (CD4, CD8, CD14, CD16, CD19, CD34, CD123, FcεRI), CD11b-FITC, CD56-FITC, CD3-BV510, CD127-BV421, CD45-Alexa Fluor 700 and CD294-PE. View supplement for more detailed information. CD45+, Lineage-, CD11b-, CD56-, CD3-, CD127+, CD294+ cells were sorted with an FACS ARIA Fusion (BD Bioscience) into 96 U bottom well plates (Corning, Amsterdam, Netherlands).
Type 2 innate lymphoid cell culture
Sorted ILC2s (100 cells/well) were expanded with human feeder PBMCs (100,000/well; 37 °C, 5% CO2) for three to five weeks. Culture medium contained RPMI 1640 Glutamax medium (Life Technologies, Darmstadt, Germany), 1% Pen/Strep (Life Technologies, Darmstadt, Germany), 10% h.i. human AB serum (Sigma-Aldrich, Taufkirchen, Germany), and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Lonza, Wakersville, USA). The medium was supplemented with 100 U/ml rh-IL-2 (Life Technologies, Darmstadt, Germany), 25 ng/ml rh-IL-4 (Miltenyi Biotech, Bergisch Gladbach, Germany), 5 µg/ml phytohemagglutinin-M (PHA-M; Sigma-Aldrich, Taufkirchen, Germany).
Cell migration assay
Migration was assessed using 5.0 µm pore size, polycarbonate membrane, polystyrene 96-transwell plates (Corning, Amsterdam, Netherlands; Hölzel, Köln, Germany) following manufacturer’s protocols. Metabolites (lower well, final conc. 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 µM, 2 µM, 3 µM and 5 µM) and cells (upper well, ≤ 100,000/well) were incubated for 6 h (37 °C, 5% CO2) in culture medium without IL-2, IL-4 and PHA. Migrated cells (25 µl) were incubated with CellTiter-Glo® Luminescence Cell Viability Assay (Promega, Mannheim, Germany) following manufacturer’s protocol. Luminescence was measured using a Tecan infinite F200 pro.
For DP2 inhibition experiments, ILC2s were incubated with fevipiprant (final conc. 0.01 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 100 nM, 1 µM, 10 µM, for 1 h, 37 °C, 5% CO2) in culture medium without IL-2, IL-4 and PHA and cell migration was measured as described above using the EC70 concentration of respective metabolites.
Cells (≤ 150,000/well) and metabolite solutions (final conc. 2.5 nM, 5 nM, 25 nM, 50 nM, 250 nM, 500 nM, 1 µM, 1.5 µM, 2.5 µM) were incubated for 24 h in culture medium without IL-2, IL-4 and PHA (U-bottom 96-well plates, 37 °C, 5% CO2). After centrifugation (5 min, 300×g, RT), the supernatant was collected and stored at − 80 °C until measurement.
For DP2 inhibition experiments, ILC2s were incubated with fevipiprant (final conc. 0.01 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 1 µM) for 1 h (37 °C, 5% CO2) and afterwards with the EC70 concentration of respective metabolites for 24 h. Cells were incubated as described above.
Supernatants were diluted 1:10 and IL-5 and IL-13 concentrations were measured by MSD immunoassays (V-PLEX Meso Dale Discovery, Rockville, MD, USA) following manufacturer’s protocol. Raw values are depicted in Additional file 1: Figs. S2 and S3.
GraphPad Prism 9.0.1 was used for analysis. Four parameter non-linear agonist or inhibitor regression models were used fit curves and to calculate EC50/IC50 values, respectively. Fitting curves constraints are indicated in respective figure legends.
Maximal PGD2-induced responses were compared with respective maximal responses of metabolites using paired One-Way ANOVA with post-hoc Dunn’s multiple comparisons tests.