THP-1 cells (ACC 16, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany) or THP-1 KO NLRP3 cells (thp-konlrp3z, Invivogen, Toulouse, France) were cultured in RPMI 1640 (11530586, Fisher scientific, Schwerte, Germany) containing 100 U/ml penicillin, 100 μg/ml streptomycin (P4333), 2 mM L-glutamine (G7513, both from Sigma-Aldrich, Taufkirchen, Germany) and 10% heat-inactivated fetal bovine serum (FBS; S0615, Sigma-Aldrich, Taufkirchen, Germany) at a density of 2–8 × 105 cells/ml. Cells were used from passage 4 to 25 and maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cell lines were regularly tested negative for mycoplasma contamination (VenorGeM Classic Mycoplasma PCR detection kit, 11–8100, Minerva Biolabs, Berlin, Germany). For generating THP-1 derived macrophages, THP-1 monocytes were seeded into 24-well plates at a density of 4 × 105cells/ml in growth medium including 25 ng/ml PMA (phorbol 12-myristate 13-acetate; tlrl-pma, Invivogen, Toulouse, France). After 48 h, adherent cells were carefully washed with PBS (phosphate buffered saline; P04-53500, Pan Biotechne, Aidenbach, Germany) and rested in PMA-free medium for 24 h. PBMCs were isolated from buffy-coat donations (Institute of Experimental Haematology and Transfusion Medicine, University Hospital Bonn) by density gradient centrifugation using Bicoll separation media (BS L6115, Bio&Sell, Nuremberg, Germany). HEK-blue IL-1β cells were cultured in Dulbecco's modified Eagle's medium (DMEM, P04-03500, Pan Biotechne, Aidenbach, Germany) containing 4.5 g/l glucose, 10% (v/v) heat-inactivated fetal bovine serum (FBS; S0615), 2 mM l-glutamine (G7513), 100 U/ml penicillin, 100 μg/ml streptomycin (P4333, all from Sigma-Aldrich, Taufkirchen, Germany), 100 μg/ml normocin, 100 μg/ml zeocin (selective antibiotics, ant-nr-1, ant-zn-1, both from Invivogen, Tolouse, France). Cells were used from passage 3 to 20 and maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. To determine if THP-1 stimulated cells secrete bioactive form of IL-1β a total of 50 μl of THP-1 supernatant was transferred to a 96-well tissue culture–treated plate and mixed with 5 × 104 IL-1β reporter cells in test media (culture media without normocin and zeocin) and incubated for 20 h at 37 °C. After IL-1β receptor stimulation of HEK-Blue cells, NF-κB and AP-1 activation-induced production of secreted embryonic alkaline phosphatase (SEAP) that can be determined with the colorimetric substrate QuantiBlue (rep-qbs, Invivogen, Toulouse, France) by reading the optical density (OD) at 620 nm.

PBMCs as well as THP-1 monocytes and differentiated THP-1 derived macrophages were preincubated with LPS from Escherichia coli 0111:B4 (tlrl-3pelps), Pam2CSK4 (tlrl-pm2s-1), Pam3CSK4 (tlrl-pms-1, all from Invivogen, Toulouse, France) or growth medium for 3 h. Afterwards PBMCs and THP-1 macrophages were stimulated for additional 3 h or THP-1 monocytes for 6 h with 300 μM BzATP (2'/3'-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate, NU-1620–25), 5 mM ATP (Adenosine 5´-triphosphate, NU-1010-100G, both from Jena Bioscience, Jena, Germany) 10 μM nigericin (4312/10, Tocris Bioscience, Bristol, United Kingdom). In selected experiments, cells were preincubated with the non-competitive P2X7 receptor antagonist A804598 (1 μM, 4473, Tocris Bioscience, Bristol United Kingdom), irreversible P2X7 receptor antagonist oxidized ATP (oxATP, 300 μM, 505758, Merck, Darmstadt, Germany), P2X4 receptor antagonist 5-BDBD (25 μM, SML0450-5MG, Sigma-Aldrich, Taufkirchen, Germany), P2X receptor antagonist PPADS (100 μM, 0625, Tocris Bioscience, Bristol, United Kingdom), NLRP3 inhibitor MCC950 (10 μM, 5479, Tocris Bioscience, Bristol, United Kingdom) or Bay 11–7082 (20 μM, B5556, Sigma-Aldrich, Taufkirchen, Germany), Caspase-1 inhibitor Ac-YVAD-cmk (40 μM, 10014, Biomol, Hamburg, Germany), pan-caspase inhibitor Z-VAD-fmk (40 μM, tlrl-vad, Invivogen, Toulouse, France), serine protease inhibitor AEBSF (300 μM, 50985.100, Biomol, Hamburg, Germany), cysteine protease inhibitor E64 (10 μM, 324890, Merck, Darmstadt, Germany), aspartic protease inhibitor pepstatin A (50 μM, 2936, Carl Roth, Karlsruhe, Germany) 1 h before stimulation. To determine the optimal concentration of caspase inhibitors required to inhibit IL-1β secretion, we incubated increasing concentrations of Ac-YVAD-cmk and Z-VAD-fmk together with nigericin (Figure S1). To examine the dependency of potassium efflux, Pam3CSK4-primed THP-1 macrophages were stimulated in the presence of potassium chloride (75 mM, 6781.3, Carl Roth, Karlsruhe, Germany). To determine the contribution of the NLRP3 inflammasome, THP1 KO NLRP3 macrophages were primed with 1 μg/ml Pam3CSK4 for 3 h followed by stimulation with BzATP, ATP or nigericin for 3 h.

After 3 h of stimulation of Pam3CSK4-primed PBMCs or THP-1 macrophages or after 6 h of stimulation of Pam3CSK4-primed THP-1 monocytes cell culture supernatants were collected and analyzed for IL-1β release using commercially available ELISA kits (88–7261-88 from Thermofisher Scientific, Darmstadt, Germany).

LDH assay was performed according to the manufacturer’s instructions (Thermofisher Scientific, Darmstadt, Germany). The percentage of LDH release was calculated compared to 100% cell lysis control.

Total RNA isolation was performed using innuPREP RNA Mini Kit 2.0 (845-KS-2040050, AnalytikJena, Jena,Germany) according to the manufacturer’s protocol. Hence, cDNA was synthesized with the help of iScript cDNA Synthesis Kit (1708891, Bio-Rad, Feldkirchen, Germany). Quantitative real-time RT-PCR (qRT-PCR) was performed as previously described [20, 21]. Primers (synthesized by TIB Molbiol, Berlin, Germany or eurofins genomics, Ebersberg, Germany) with the following sequences were used: GAPDH, 5’-CTCTCTGCTCCTCCTGTTCGAC-3’ and 5’-TGAGCGATGTGGCTCGGCT-3’; IL1B, 5’-TGGAGCAACAAGTGGTGT-3’ and 5’-TTGGGATCTACACTCTCCAGC-3’; IL18, 5´-TGCCAACTCTGGCTGCTAAA-3´ and 5´-TTGTTGCGAGAGAAGCGAT-3´, PRTN3, 5´- GCCGGCCACATAACATTTGC-3´and 5´-TACCCGCGTGAAGAAGTCAGG-3´; ELANE, 5´-AACGGCTACGACCCCGTAAA-3´ and 5´-CTGCACGTTGGCGTTGATGG-3´; CTSG, 5´-GCTGAGGCAGGGGAGATCATCG-3´ and 5´-GGGTGTTTTCCCGTCTCTGGA-3´

Fold difference in gene expression was normalized to the housekeeping gene GAPDH showing the most constant expression levels. The reaction mix containing cDNA template, primers and SYBR Green (iTaq Universal SYBR Green Supermix; 172–5125, Bio-Rad) was run under the conditions as previously described.

Data are expressed as means + SEM. For multiple comparisons, statistically significant differences were determined by one-way ANOVA followed by a Dunnett´s or Tukey´s post-test and considered significant at *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. For studies of inhibitory effects ATP, BzATP or nigericin induced IL-1β release was set to 100%. All other values were calculated accordingly. Statistical differences were assessed by one-sample t test against 100%. Statistical analysis was performed using GraphPad Prism software.