PBMCs were counted and resuspended in Dutch modified Roswell Park Memorial Institute (RPMI) medium (Life Technologies), supplemented with 50 µg/mL gentamicin (Thermo Fisher Scientific), 1 mM sodium pyruvate (Thermo Fisher Scientific), and 2 mM Glutamax (Thermo Fisher Scientific). 5 x 105 PBMCs were cultured in a final volume of 200 μL/well in round bottom 96-well plates (Greiner Bio-one) and stimulated with of heat-inactivated SARS-CoV-2 Wuhan-Hu-1 virus variant (1.19 x 104 TCID50/ml; NRW-42 isolate, kindly provided by Heiner Schaal, University Hospital Düsseldorf, Germany) (17 (link)), heat-inactivated Staphylococcus aureus (1 x 106 CFU/mL; clinical isolate), heat-inactivated influenza virus H1N1 (3.2 x 105 K/mL, kindly provided by Ortwin Adams, University Hospital Düsseldorf, Germany), Imiquimod/R837 (10 µg/ml; InvivoGen), Poly (I:C) (10 µg/mL; InvivoGen), R848 (10 µg/mL; InvivoGen), purified lipopolysaccharide (LPS) derived from Escherichia coli O55:B5 (10 ng/mL; Sigma-Aldrich), or recombinant human interleukin-1 alpha (IL-1α) (10 ng/mL; R&D Systems), in the presence of dexamethasone (10 nM or otherwise stated; Sigma-Aldrich) or vehicle control (dimethyl sulfoxide; DMSO). After 4h cells were lysed in RLT buffer (Qiagen) and stored at −80°C for subsequent RNA isolation. After 24 h or 7 days, supernatants were collected and stored at –80°C until analysis.
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