Mcc950

Thirty mice were assigned randomly into five groups (n = 6 each): (1) phosphate-buffered saline control (Con); (2) MCC950 control (MCC); (3) OVAlbumin- (OVA-) induced lung allergic inflammatory group (OVA); (4) OVA group treated with sevoflurane (OVA + SVF); and (5) OVA group treated with MCC950 (OVA + MCC).
OVA sensitization was performed by intraperitoneal (i.p.) injection of 10 μg of OVA (Sigma, St. Louis, MO, USA) adsorbed to 1 mg of alum (potassium aluminium sulphate, Sangon Biotech, Shanghai, China) on day 0. Fourteen days later, the animals were challenged by 1% OVA solution for 30 min for 7 consecutive days. The control mice were sensitized and challenged with saline alone. Immediately after OVA provocation, the mice received 3% sevoflurane (Maruishi Pharmaceutical, Osaka, Japan) for 1 h from day 14 to 20. MCC950 (10 mg/kg, i.p.) (Abmole Bioscience Inc., USA), a recently-described small molecule inhibitor of the NLRP3 inflammasome, was administered every two days postchallenge with OVA for comparison. At 24 h after the final challenge, the mice were sacrificed, and bronchoalveolar lavage fluid (BALF) was collected. Half of each lung was stored at −80°C for Western blotting, and the other half was fixed overnight in buffered 4% formaldehyde solution for histology analysis.

THP-1s (TIB-202; American Type Culture Collection) were maintained in RPMI supplemented with 10% (vol/vol) heat-inactivated FBS, 0.05 nM β-mercaptoethanol, 100 IU/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified incubator. Two days before experimentation, the cells were replated in media without antibiotics in a 48-well plate at a concentration of 2 × 10⁵ cells/well and incubated with phorbol 12-myristate 13-acetate (PMA) for 24 hours to allow differentiation into macrophages. Macrophages were primed with 100 ng/mL Pam3CSK4 (Invivogen) for 16 hours prior to bacterial infections or anthrax toxin treatments. For experiments involving LPS, cells were pretreated with 500 ng/mL LPS (Sigma-Aldrich) for 3 hours. For experiments involving Nigericin, cells were treated with 10 μM Nigericin (EMD Millipore) for 6 hours. For experiments involving MCC950, cells were treated with 1 μM MCC950 (Sigma Aldrich) 1 hour prior to infection.

Alexa Fluor™ 488 Protein Labelling Kit, E. coli BL21 Star™ (DE3) strain, isolectin GS-IB₄ from Griffonia simplicifolia conjugated with Alexa Fluor488 or Alexa Fluor568, IL-18 (Rat) ELISA kit, pHrodo™ red E.coli BioParticles conjugate, were purchased from Invitrogen, ThermoFisher Scientific (USA). Cell culture reagents DMEM Glutamax, fetal bovine serum, horse serum, penicillin–streptomycin, Versene (1:5000) solution were from Gibco, ThermoFisher Scientific (USA). Poly-(l)-lysine was from R&D systems (USA), IL-1β (Rat) ELISA kit from Abbexa (United Kingdom), FamFlica Caspase-1 Assay Kit from ImmunoChemistry Technologies (USA), anti-TLR4 antibody from Santa Cruz Biotechnology (USA). Cell permeable Ac-YVAD-CHO, VX-765, GSK2795039, MCC950, and IgG were obtained from Merck (Germany). All other materials were purchased from Sigma-Aldrich (USA).

Culture medium of differentiated macrophages was exchanged after 5 days of incubation. For inhibitor experiments, cells were preincubated with either 0.1% DMSO (Merck, KGaA, Darmstadt, Germany), MCC950 (10 μM) (Merck), KINK‐1 (5 μM) (Merck), MitoTEMPO (10 μM) (Merck), entospletinib (5 μM) (Gilead Sciences, Inc., Foster City, CA, USA), or tamatinib/R406 (5 μM) (InvivoGgen, San Diego, CA, USA) in RPMI medium for 2 h at 37°C and 5% CO₂. Thereafter or without inhibitors, cells were stimulated with lipopolysaccharide (LPS) (5 μg/ml) (Merck) or SARS‐CoV‐2 spike protein (0.1 μg/ml) and incubated for 4 h at 37°C and 5% CO₂ to prime the inflammasome process. Inflammasome activation and IL‐1β secretion were initiated by addition of nigericin (5 μM) (Merck) or ATP (5 mM) (Thermo Fisher) and the cells were further incubated for 2 h (Theobald et al, 2021 (link)).

All LPS variants were
purchased from Innaxon. Unless otherwise indicated, cells were stimulated
with 100 ng/mL ultrapure Smooth-LPS from Salmonella
minnesota (S-LPS) for 18 h. Rough-LPS R595 (Re) from S. minnesota (R-LPS) was used at 100 ng/mL, while
MPLA R595 (Re) from S. minnesota (MPLA)
was used at 1 μg/mL. For TLR2 activation, PAM2CSK4 (Invivogen)
was added at 10 ng/mL for 18 h. hIL-1β (Merk) was used as control
for NF-κB activation and added at a final concentration of 100
ng/mL. FP11, FP111, and FP18 compounds were resuspended in ultrapure
DMSO and diluted in culture medium. Anti-human IFNAR2 neutralizing
antibody (clone MMHAR-20) was purchased from PBL Assay Science and
used at 1 μg/mL. The NLRP3 inhibitor MCC950 was purchased from
Merck and added to cells at the following concentrations: 0.01, 0.1,
1, and 10 μM.

The Smac-mimetic compound A and the caspase inhibitor IDN-6556 (Idun Pharmaceuticals) were synthesised by TetraLogic Pharmaceuticals. The RIPK3 inhibitor GSK’872 was from Calbiochem. The TAK1 inhibitor (5Z)-7-oxozeaenol was from Tocris Bioscience. Cycloheximide and nigericin were from Sigma. The NLRP3 inhibitor MCC950 was purchased from Merck. Caspase-1-specific inhibitor Vx765 was from MedChemExpress. Ultrapure LPS-EB, poly(I:C), P₃Cys, and CpG were purchased from Invivogen.

Rifampicin was purchased from AppliChem (Darmstadt, Germany). CHIR99021, dimethyl sulfoxide, MCC950, MitoTEMPO, Pifithrin-α, and VX-765 were purchased from Merck (Darmstadt, Germany). CsA was obtained from Cayman Chemicals (Ann Arbor, USA). BTP15 was synthetized as described recently [38 (link)].