Reactants

Phorbol 12-myristate-13-acetate (PMA) (P8139-MG, Sigma Chemical Co., St. Louis, MO, USA), dexketoprofen (sc-357330, Santa Cruz Biotechnology, Santa Cruz, CA, USA), lipopolysaccharide (LPS) (L4391, Sigma Chemical Co., St. Louis, MO, USA), nigericin (sc-201518B, Santa Cruz, CA, USA), adenosine triphosphate (ATP) (sc-202040A, Santa Cruz, CA, USA), bovine serum albumin (BSA) (9048–46-8, Sigma Chemical Co., St. Louis, MO, USA), DAPI (Santa Cruz Biotechnology, Santa Cruz, CA, USA), saponin (47,036, Sigma Chemical Co., St. Louis, MO, USA), bioBLUPrestainesProtein loader (GTPBM0003, Biorad Laboratories Inc., Hercules, CA, USA). Primary antibodies: gasdermin D (ab219800, Abcam, Cambridge, UK), IL-1β (ab243091, Abcam, Cambridge, UK), caspase-1 (NBP1-45,433, Novus Biologicals, Colorado, USA), GADPH (5174S, Cell Signaling, Danvers, MA, USA), NLRP3 (NBP2-12,446, Novus Biologicals, Colorado, USA) and ASC (13833S, Cell Signaling, Danvers, MA, USA). Secondary antibodies: rabbit (401,353-2ML, Burlington, MA, USA) and mouse (401,253-2ML, MilliporeSigma, Burlington, MA, USA).

Cell culture

Cells were incubated at 37 °C in a 5% CO2 atmosphere. Primary human THP1 monocytes (TIB-202, ATCC, Manassas, VA, USA) were cultured in RPMI-1640 medium (Gibco, Invitrogen, Eugene, OR, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, Eugene, OR, USA) and 1% antibiotics solution (L0010-100, Nuaillé, France). Macrophages were obtained from THP1 cells differentiation, PMA 50 ng/mL 24 h.

Macrophage cells were treated with dexketoprofen (1 nM or 100 nM for 15 min) and then primed with LPS 1 µg/µL 4 h and stimulated with ATP 5 mM or nigericin 10 µM for 30 min. Cells were scrapped and centrifuged for 5 min at 1200g. Supernatant and pellet was stored at – 20 °C for further assays.

Protein extraction

Pellet cells were lysed using RIPA (Thermo Fisher Scientific, MA, USA) with PMSF (Thermo Fisher Scientific, MA, USA) 1 nM as lysis buffer. Cells were resuspended in lysis buffer and kept in ice for 30 min and centrifuged for 5 min at 12.000g and the supernatant was collected. Protein quantification was done using the BCA kit (Thermo Fisher Scientific, MA, USA). Then, protein aliquots 10 ng/µL were mixed with NuPAGE LDS sample buffer (Invitrogen Eugene, OR, USA) and subjected to thermal shock for 5 min at 95 °C.

Electrophoresis

Samples were loaded in mini-PROTEAN TGX precast gels 4–20% (4,561,096, Biorad Laboratories Inc., Hercules, CA, USA) and ran in Tris/glycine/SDS buffer (1,610,772, Biorad Laboratories Inc., Hercules, CA, USA) 45′ 200 V.

Inmunoblot

Proteins were transferred from gel 0.45 µm (1,620,115, Biorad Laboratories Inc., Hercules, CA, USA) using a TransBlot Turbo (Biorad Laboratories Inc., Hercules, CA, USA). Membranes were blocked by immersion in a BSA 5% solution during 1 h with gentle agitation. Primary antibodies 1:1.000 were incubated overnight at 4 °C. Membranes were washed three times with PBS–Tween20 solution and incubated with secondary antibody 1:10.000 for 1 h at room temperature. Membranes were washed three times and developed in a ChemiDoc MP Imaging System (Biorad Laboratories Inc., Hercules, CA, USA) using WesternBright Sirius detection kit (Advansta, San Jose, CA, USA).

Lactate dehydrogenase (LDH) activity assay

LDH activity was measured using the LDH Assay Kit/Lactate Dehydrogenase Assay Kit (Colorimetric) (ab102526, Abcam, Cambridge, UK) following the recommended procedure. In a 96-well plate, supernatant samples and reaction mix were added. The plate was analyzed every 2–3 min for at least 30 min with iMark microplate reader (Biorad Laboratories Inc., Hercules, CA, USA) in kinetic mode at 37 °C.

ELISA (enzyme-linked immunosorbent assay)

Supernatant IL-1β levels were analyzed using human IL-1 beta ELISA Kit (ab214025, Abcam, Cambridge, UK) and following the recommended procedure. Samples and antibody cocktail were added to each well and incubated for 2 h at room temperature. Each well was washed three times and the development solution was added. After 10 min incubation in the dark, stop solution was added and OD at 450 nm was record using an iMark microplate reader.

Immunofluorescence assay

Macrophages were grown on 1 mm-width glass coverslips in RPMI-1640 medium containing 10% FBS and 1% antibiotics. After inflammasome treatment, cells were washed twice with PBS, fixed in 10% paraformaldehyde for 10 min at room temperature, washed three times with PBS and permeabilized with 0.1% saponin in PBS for 30 min at room temperature and incubated in blocking buffer (BSA 1% in PBS) for 1 h. After blocking, cells were incubated overnight at 4 °C with a 1:100 primary antibody solution in blocking buffer. Cells were washed three times with PBS and incubated with a 1 µg/ml DAPI solution in PBS for 5 min and washed five times with PBS. Glass coverslips were mounted on microscope slides using Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA) and visualized using a DeltaVision microscope (imsol, Preston, UK).

Docking study

Dexketoprofen mol2 and human NLRP3 pdb file and file were used for molecule–protein interaction study. Dexketoprofen mol2 structure file was designed using GaussView 5.08 software. Atoms’ distance, angles and polar charges were optimized using Gaussian09W software. Human NLRP3 pdb file was obtained as 6NPY file from RCSB Protein Data Bank. NEK7 structure was removed from 6NPY file using UCSF Chimera 1.16 software before docking analysis. Polar hydrogens and Gasteiger charges were added using UCSF Chimera 1.16 software. Autodock Vina software was used for docking validation.

For human NLRP3 (PDB:6NPY), a grid box was made with x, y, z dimensions of 37 Å, 32 Å and 36 Å centered with x, y, z coordinates of 93.177 Å, 96.763 Å and 81.168 Å. Non-polar hydrogens and lone pairs were removed, water molecules and chains of non-standard residues were ignored and all non-standard residues were not ignored for the human NLRP3 (PDB:6NPY) receptor. Non-polar hydrogens and lone pairs were not removed for the dexketoprofen Ligand. Docking analysis was done based on maximum exhaustiveness of search and maximum energy difference of 2 kcal/mol. Ten binding modes were given after analysis which were visualized and analyzed using UCSF Chimera 1.16 software. Interaction type and distance between dexketoprofen atoms and residues were analyzed using structure measurements tool provided by UCSF Chimera 1.16 software.

ATPase assay

Purified recombinant human NLRP3 (Novus Biologicals) was incubated at 37 °C with dexketoprofen and MCC950 10 mM for 15 min in the reaction buffer. ATP (250 μM, Ultra-Pure ATP) was then added and the mixture was further incubated at 37 °C for another 40 min. The amount of ATP converted into adenosine diphosphate (ADP) was determined by luminescent ADP detection with ADP-Glo Kinase Assay Kit (Promega, Madison, MI, USA) according to the manufacturer’s protocol. The results were expressed normalized to the enzyme activity of NLRP3 treated with vehicle.

Luminescence was measured using a Typhoon FLA 9500 Biomolecular Imager (Molecular Dynamics, Sunnyvale, CA, USA) and data were processed using ImageQuant™ software.

Statistics

All data are expressed as means ± SEM. Statistical differences among the different groups were measured using an unpaired Student’s t test. A P value of ≤ 0.05 was considered statistically significant. Statistical analyses were performed using Prism software version 5.0a (GraphPad, San Diego, CA). Asterisks in the figures represent the following: * P ≤ 0.05; ** P ≤ 0.01; and *** P ≤ 0.001.