Phorbol myristate acetate (pma)

Human sputum cell or PBMCs samples were labeled without stimulation for flow cytometry with specific antibodies using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to manufacturer’s instructions.
For flow cytometric analyses of the murine samples, staining for transcription factors was performed by using Foxp3 Staining Buffer Set (eBiosciences), while cytokine staining was performed by using the fixation/permeabilisation solution Kit (BD Bioscience Cytofix/Cytoperm™) according to the manufacturer’s protocols. For intracellular cytokine staining, cells were stimulated for 4h with 50 ng/ml PMA (Applichem), 500 ng/ml Ionomycin (Thermo Fisher Scientific) and 1:1000 GolgiPlug (BD Bioscience). Flow cytometric analysis was performed using a BD LSRII Fortessa flow cytometer (BD Bioscience). Flow cytometric data of both human and mouse samples were analyzed with FlowJo software (FlowJo). For gating strategy of human sputum cells (0.5x10⁶ cells) or PBMCs (2.0x10⁶ cells) samples see Figures 5A, S2B; the murine gating strategy is shown in Figure S1. Antibodies used for flow cytometry are listed in Table S2 (murine) and Table S6 (human).

U937 monocytes at a concentration of 1 x 10⁶ cells/ml were differentiated into macrophage-like cells by incubating with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Applichem, Germany) in RPMI complete medium for 24 hours at 37 °C and 5 % CO₂. Cells were washed twice with complete medium and then rested for a further 48 hours in fresh PMA‐free complete medium to obtain resting macrophages (Sproston et al., 2018[42 (link)]; Rios de la Rosa et al., 2017[37 ]).