Materials

Pharmacological reagents were obtained from the following manufacturers: Sigma (celecoxib, ibuprofen, diclofenac, flufenamic acid, meclofenamic acid, mefenamic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), DIDS, 4-(2-Butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB), Lipopolysaccharide from Escherichia coli O26:B6, ATP and nigericin), Merck-Millipore (Ac-YVAD-CHO), Acros organics (MNS) and Invivogen (MSU crystals, ultrapure flagellin from Salmonella typhimurium). All other materials/reagents were obtained from Sigma unless otherwise stated.

Cell culture and assays

iBMDMs41 were obtained from Clare Bryant (Department of Veterinary Medicine, University of Cambridge). iBMDMs were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), 10% fetal bovine serum (FBS, Life Technologies), 100 U ml−1 penicillin and 100 μg ml−1 streptomycin (PenStrep). Cells were seeded overnight at 5 × 105 ml−1 in 24- or 96-well plates. Cells were primed with LPS then treated with drug or vehicle (DMSO) in serum-free media for 15 min. Following drug incubation, inflammasomes were stimulated by adding ATP (5 mM) for 1 h. Supernatants were removed and analysed for IL-1β content by ELISA (DuoSet, R&D systems) according to manufacturer’s instructions. Primary mouse BMDMs were isolated by flushing marrow from femurs and tibias of wild-type C57BL/6 (Charles River) or NLRP3−/− mice and cultured with L929 mouse fibroblast supernatant-conditioned media for 7–10 days. Human THP-1 peripheral blood monocyte-like cells were cultured in RPMI-1640 Medium supplemented with 10% FBS, 100 U ml−1, 100 μg ml−1 PenStrep, 20 mM l-Glutamine and 55 μM 2-mercaptoethanol (Life Technologies). Following priming for 4 h with LPS and 15 min pre-treatment with drug in serum-free media, inflammasomes were activated with nigericin (10 μM) for 1 h, MSU crystals (250 μg ml−1) for 4 h, transfected ultrapure flagellin from Salmonella typhimurium (100 ng per 100,000 cells) for 2 h, or transfected DNA (pEF/V5-His A plasmid empty vector (Life Technologies, 66.7 ng per 100 K cells)) for 4 h. Transfections described above were performed using Lipofectamine 3000 (Life Technologies) according to manufacturer’s instructions.

Western blotting

Western blot analysis was performed on supernatants for IL-1β and caspase-1. Samples were run on 12% (IL-1β) or 15% (caspase-1) SDS polyacrylamide gels. For caspase-1 blots THP-1 supernatants were concentrated before preparation and loading by centrifugal filtering (Amicon 10 K centrifugal filters) according to manufacturer’s instructions. Gels were transferred at 15 V onto nitrocellulose membrane (GE Life Sciences) using a Trans-Blot s.d. semi-dry transfer system (Bio-Rad) before blocking with 5% w/v milk in phosphate-buffered saline, 1% Tween 20 (Sigma) (PBST) for 1 h at room temperature. Membranes were washed and incubated (4 °C) overnight in goat anti-mouse IL-1β (100 ng ml−1, R&D Systems) or rabbit anti-human caspase-1 p10 (666.6 ng ml−1, Santa Cruz) primary antibodies in PBST, 0.1% bovine serum albumin (BSA). Following this, membranes were washed and incubated with rabbit anti-goat (550 ng ml−1) or goat anti-rabbit IgG (250 μg ml−1, Dako) in 5% milk in PBST for 1 h at room temperature. Finally, membranes were washed and incubated in Amersham ECL Western Blotting Detection Reagent (GE Life Sciences) before exposure on photographic film (Scientific Laboratory Supplies). Uncropped blots are presented in Supplementary Fig. 12.

Cell death assays

Cell death was measured by assessing lactate dehydrogenase release using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega) according to manufacturer’s instructions.

ASC speck imaging

Live imaging of ASC speck formation was performed using iBMDMs transfected to stably express ASC conjugated to mCherry protein. Mouse ASC (Accession Number NM_023258) was amplified by PCR using primers flanked by gateway recombination sequences (forward primer: 5′- ATGGGGCGGGCACGAGATGCC -3′, reverse primer: 5′- GCTCTGCTCCAGGTCCATCAC -3′). A third generation ‘pLNT’ lentiviral transfer system was used to express N-terminally mCherry-tagged amplified ASC gene from a constitutive ubiquitin-ligase C promoter42. Stably transduced cells were plated overnight at 5 × 105 cells per ml. The following day, cells were primed with LPS (1 μg ml−1, 2 h). 1 h into priming, Hoechst 33342 (2 μg ml−1, Immunochemistry) was added to aid identification of the cells. Following priming, media was changed to DMEM containing 25 mM HEPES pH 7.4 and cells transferred to a BD Pathway Bioimager 855 (BD Biosciences) and imaged at 37 °C. Cells were pre-treated with drug or vehicle for 15 min before imaging. Images were acquired using a × 20/0.75 UApo objective and the following filter setups: Hoechst Ex. 360/10 Di. Em. 84101; mCherry Ex. 555/28 Di. Em. 84101. Images were collected and a montage of 3 × 3 was created. Following drug incubation, cells were stimulated by addition of ATP (5 mM) directly into the well and imaged as described for 30 min. Specks were quantified by blind-counting nine separate fields of view (1.2 mm2) using ImageJ.

Cysteine modification and NMR

Flufenamic acid or MNS (0.05 mmol) was dissolved in DMSO-d 6 (400 μl) in an NMR tube. N-Acetyl-L-cysteine methyl ester (17.7 mg, 0.10 mmol) solubilized in DMSO-d 6 (100 μl) was added and an NMR spectrum was recorded every 10 min after the addition for 2 h and then every 1 h until 24 h. A Bruker Avance 400 spectrometer was used to record 1H spectra at 300.1 MHz. Chemical shifts are defined in parts per million and referenced against tetramethylsilane (δ=0). NMR analysis was conducted using Bruker’s TopSpin software package.

Electrophysiological recordings

Membrane currents were measured using the whole-cell configuration of the patch-clamp technique. An EPC-10 patch-clamp amplifier (HEKA) was interfaced to a computer for pulse application and data recording using the programme PatchMaster (HEKA). Patch electrodes of 3-5 MΩ were fabricated on a two-stage puller (Narishige PC-10) from borosilicate glass (Hilgenberg). For all electrophysiological experiments, 105 iBMDM cells were seeded in 24-well plates on glass coverslips and primed the next day with 1 μg ml−1 LPS for 4 h. For electrophysiological recordings of ATP-induced cation currents, patch electrodes were filled with an intracellular solution containing (in mM): KCl, 120; HEPES, 10; EGTA, 11; CaCl 2 , 1; MgCl 2 , 2 (pH 7.3). LPS-primed iBMDM cells were superfused with extracellular solution containing (in mM): NaCl, 130; KCl, 5; HEPES, 10; D-glucose, 10; CaCl 2 , 2; MgCl 2 , 1 (pH 7.4). The effect of flufenamic/mefenamic acid was investigated after a stable cation current was induced by application of 5 mM ATP. DMSO was used as vehicle control in these experiments. Leak-subtracted currents were analysed at +90 mV and data are presented as mean values±s.e.m. For volume-regulated Cl− current recordings, patch electrodes were filled with the following intracellular solution (in mM): N-Methyl-D-Glucamine-Chloride (NMG-Cl), 120; HEPES, 10; EGTA, 11; CaCl 2 , 1; MgCl 2 , 2; Na 2 ATP, 3 (pH 7.3). Cells were kept in iso-osmolar extracellular solution containing (in mM): NMG-Cl, 50; HEPES, 10; D-glucose, 10; CaCl 2 , 2; MgCl 2 , 1; D-mannitol, 170 (300 mosmol per kg, pH 7.3). To activate VRAC currents, superfusion was changed to hypo-osmolar extracellular solution containing no D-mannitol (130 mosmol per kg, pH 7.3). To permit a rapid exchange of solutions for drug application, cells were continuously superfused using a four-barrel microperfusion pipette positioned near the recorded cell. Extracellular solutions containing the indicated NSAID were applied after stable activation of Cl− currents; DMSO was used as vehicle control. All recordings were made at a temperature of 23–26 °C. Whole-cell currents were filtered at 3 kHz and stored for subsequent analyses. Analyses of leak-subtracted currents at +90 mV were performed with the programme FitMaster (HEKA).

Fluorescence imaging

105 iBMDMs were seeded one day before experiments on glass coverslips in 24-well plates and primed with LPS (1 μg ml−1, 4 h) before fluorescence imaging experiments. Subsequently cells were loaded with fluorescent dyes in Na+/K+ containing extracellular solution at room temperature (20–23 °C): for Ca2+ imaging experiments with 3 μM fura-2-acetoxymethylester (Fura-2-AM) for 30 min, for Na+ imaging experiments with 10 μM sodium-binding benzofuran-isophthalate acetoxymethyl ester (SBFI-AM; both dyes from Molecular Probes) for 60 min. After washing with extracellular solution, glass coverslips were mounted in a chamber on an inverted Olympus IX50 microscope equipped with a × 40 water immersion objective UApo/340 (Olympus Optical, Co.). The fluorescence imaging system consisted of a monochromator, a charge-coupled device (CCD) camera and a Windows 7 based image processing software (Till Photonics). Cells were exposed to alternating 340±5 and 380±5 nm wavelengths of UV light and emission light was passed through a 400 nm dichroic mirror and a 420 nm long pass emission filter (both Olympus) before image acquisition by the CCD camera. Images were collected every 20 s. Cells were continuously superfused with Na+/K+-containing extracellular solution using a four-barrel microperfusion pipette positioned in close proximity to the viewing field. Ca2+ and Na+ influx was induced by extracellular solution containing 5 mM ATP in the presence or absence of mefenamic acid. DMSO was used as vehicle control in all experiments. For each individual cell, mean intensity values from background subtracted pictures were determined and ratios F340/F380 calculated, accordingly. Data are presented as mean values±s.e.m.

Regulatory volume decrease

THP-1 cells were adjusted to a density of 1 × 106 cells per ml in RPMI media (10% FBS, 1% P/S, 1% Glutamine) and primed with LPS (1 μg ml−1, 4 h). A 300 mOsm isotonic buffer was prepared consisting of 147 mM NaCl, 10 mM HEPES, 13 mM glucose, 2 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , adjusted to pH 7.4 using NaOH. From this a 90 mOsm hypotonic buffer was achieved by diluting the isotonic buffer 1:4 with sterile water. Cell size and viability measurements were performed on a BD FACSVerse flow cytometer (BD). THP-1 cells were incubated in a 37 °C water bath and a series of measurements were taken for 60 min with 10,000 events recorded each run. Cell swelling was initiated by aspiration of isotonic buffer followed by addition of hypotonic buffer containing respective drugs. Cell volume measurements by forward scatter width were normalized against the average cell volume before hypotonic stimulus.

Animal experiments

Animals were maintained under standard laboratory conditions: ambient temperatures of 21 °C (±2 °C), humidity of 40–50%, 12 h light cycle, ad libitum access to water and standard rodent chow. All surgeries were performed with the surgeon concealed to the treatment and/or genotype, and all behavioural and histological analyses were performed by a blinded observer. Treatments were randomly allocated. All animal experiments were carried out in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986 and approved by the Home Office and the local Animal Ethical Review Group, University of Manchester.

Air pouch inflammation model

The air pouch model was used to assess the NLRP3-dependent response to MSU crystals23. On day 0 a subcutaneous air pouch was raised in the dorsum of male C57BL/6 mice (30–35 g) by the injection of 4 ml sterile air (filtered through 0.22 μm pore size) with a 25 gauge needle. This was repeated on day 3. On day 7, pouches were injected with 1 ml MSU crystals (3 mg ml−1 in sterile saline) or vehicle with or without flufenamic acid (20 mg kg−1 in sterile saline, 5% Cremaphor EL, 5% DMSO) or vehicle. Following 6 h incubation, mice were killed by raising the concentration of CO 2 and pouches were lavaged by injecting 4 ml PBS, 1% BSA, 5 mM EDTA. Lavage was collected from each animal, passed through 100 μm cell strainers before analysis by ELISA as above or by flow cytometry. For the flow cytometry cells were adjusted to a density of 5 × 106 cells per ml in ice-cold PBS, 1% BSA, 5 mM EDTA before plating out on a clear V-bottomed 96-well plate (Thermo Scientific) at 200 μl per well. Cells were stained with antibodies (Anti-CD45 conjugated to FITC at 1 ng ml−1, anti-Ly6G conjugated to APC at 1 ng ml−1 and anti-F4/80 conjugated to PE at 2.5 ng ml−1, all Tonbo Biosciences) for 45 min on ice in the dark before washing twice and fixing in paraformaldehyde (3.7–4.1% in PBS, 200 μl) for 15 min at room temperature. The following day, cells were analysed on a FACSVerse flow cytometer (BD Biosciences) with BD FACSuite software.

Peritoneal inflammation model

Randomly allocated wild-type (C57BL/6) and NLRP3 KO mice (male, 30–35 g) were dosed i.p. with mefenamic acid (50 mg kg−1, 25% (w/v) (2-hydroxy)propyl-β-cyclodextrin in PBS solubilized with 0.1 M NaOH then pH adjusted to 7.4), cyclodextrin vehicle or the selective NLRP3 inhibitor MCC950 (50 mg kg−1, PBS) and LPS (1 μg in 0.5 ml, PBS) for 4 h. Following LPS prime, mice were anaesthetized with isoflurane (induced at 3–4% in 33% O 2 , 67% NO 2, maintained at 1–2% whilst kept at 37 °C on a heat blanket) before injection with mefenamic acid, MCC950 or vehicle as before and ATP (0.5 ml, 100 mM, PBS) or PBS for 15 min. Peritoneums were lavaged and plasma taken by cardiac puncture before analysis by ELISA for IL-1β and IL-1α.

Aβ-induced memory deficit

Female Lister Hooded rats initially weighing 200–230 g (n=5–10) were used for the following study. Soluble Aβ 1–42 oligomers were prepared at a concentration of 0.5 mM in sterile 0.1 M phosphate buffer saline as previously described29,43. Rats were anaesthetized with isoflurane (induction at 4% in O 2 and maintained at 2% in O 2 ) and placed on a stereotaxic frame. Aβ oligomers were injected in the left ventricle (bregma: −0.8 mm anteroposterior, −1.5 mm lateral, −4.5 mm dorsoventral at a rate of 2.5 μl min−1; final volume: 10 μl, 5 nmol. Rats underwent surgery (vehicle or Aβ 1–42 oligomers) on day 0. Rats were treated with daily intraperitoneal (i.p.) injections of vehicle or mefenamic acid (5 mg kg−1, 25% (w/v) (2-hydroxy)propyl-β-cyclodextrin in saline solubilized with 0.1 M NaOH then the pH was adjusted to 7.4) from day −1 to day 13. We estimated based on previous literature that the dose of mefenamic acid administered here was likely to be efficacious against the inflammasome44,45. Treatment was then stopped until the last day of testing (day 35).

Transgenic mouse model of Alzheimer’s disease

Male 3xTgAD mice expressing mutant PS1M146V, APPSwe, TauP301L, and control wild-type (129/C57BL6) mice were originally supplied by Frank LaFerla (Irvine, CA, USA)46. At 13–14 months old 3 × TgAD (n=20) and wild-type (n=20) mice were randomly allocated into mefenamic acid- or vehicle- (60% DMSO, 40% Cremophor A25 Sigma Aldrich) treated groups. The animals were anaesthetized with 2 to 5% isoflurane (30% O 2 and 70% N 2 O) and implanted with ALZET 200 μl minipump. After four days of priming in the animal, the minipumps reached a sustained administration rate of approximately 25 mg kg−1 day−1 for 28 days. 36 days after minipump implantation the animals were terminally anaesthetized with 2 to 5% isoflurane (30% O 2 and 70% N 2 O) and then transcardially perfused with 0.9% saline. The brains were removed and fixed by immersion in 4% paraformaldehyde in 0.2 M phosphate buffer for 24 h followed by a 24 h immersion in 30% sucrose/PBS and then frozen on dry ice. The brains were stored at −80 °C until analysis. One mouse died during minipump implantation.

Novel object recognition

NOR was performed as previously described47,48. Briefly, the rats were placed in the NOR box (52 × 52 × 31 cm) and left free to explore two copies of the same object for 3 min (Acquisition phase). Animals were taken out of the box for an inter-trial interval (2 min) then placed back in the same box for a further 3 min, then presented with an identical copy of the previous object and a novel object (Retention phase). Both sessions are recorded on video and the time spent exploring each object was scored. The discrimination index is calculated as (novel−familiar)/(novel+familiar). Rats were tested in the NOR task on day 14 and day 35 after surgery. NOR was also performed using the 3 × TgAD transgenic mice with a white Perspex circular arena 30 cm in diameter, 8 min acquisition phase, 1 h inter-trial interval and a 4 min retention phase. This was performed 18 days after osmotic minipump implantation. Three mice were excluded as they failed to explore for at least 4 s.

Immunohistochemistry

Free-floating serial 30 μm sections were taken from the wild-type and 3 × TgAD mice using a microtome (Bright Instruments Ltd., UK) and stored in cryoprotectant (30% ethylene glycol, 20% glycerol in 0.2 M phosphate buffer). Sections were washed (3 × ) in PBS for 5 min and blocked for 1 h in 10% normal donkey serum (NDS) in 0.3% Triton X-100 PBS (PBST). This was followed by an overnight incubation with primary antibody in 2% NDS in PBST at 4 °C. Specificity controls were performed on additional 3 × TgAD sections with omission of the primary antibodies. Sections were then washed (3 × ) in PBST and then incubated in with Alexa-488 (visualizing Iba1) and Alexa-594 (visualizing IL-1β) conjugated secondary antibodies (Invitrogen) at 1:500 dilution in 2% NDS in PBST for 2 h. Sections were then washed (3 × ) in PBST and mounted using ProLong Gold Antifade Mountant with DAPI (Thermo Fischer Scientific, Inc., USA). Primary antibodies used were anti-Iba1 (1:1000, Wako Ltd) and anti- IL-1β (1:200, R&D Systems).

Microscopy and quantification of microglial activation

Images were collected on an Olympus BX51 upright microscope using a × 40 objective and captured using a Coolsnap ES camera. High power field images were taken of the subiculum, CA1 region of the hippocampus and the outer cortex at bregma −2.3 mm, −2.6 mm and −2.9 mm as shown in Supplementary Fig. 10a. These regions were chosen based on previous publications on pathology progression in 3 × TgAD mice30. Total microglia were counted and the percentage of IL-1β positive microglia recorded. Microglial morphologies were scored on an activation scale of 0 to 3 based on microglia categories previously described31. Scores of 0–1 were considered resting, and 2–3 considered activated (Supplementary Fig. 10b). Each section was treated as a technical replicate, as such scores were averaged for each animal. Counting and scoring were performed by a blinded observer. Example images were collected on a Leica TCS SP5 AOBS upright confocal using a × 63 objective and × 2 confocal zoom. Images were overlaid and stacked using Fiji Image J49.

Statistical analyses

Data are presented as mean values+standard error of the mean (s.e.m). Statistical analyses performed were one-sample t-tests, one-way analysis of variance (ANOVA) and two-way ANOVA tests with Sidak corrected post hoc. Equal variance and normality were assessed with the Levene’s test and the Shapiro–wilk test, respectively, and appropriate transformations were applied when necessary. Accepted levels of significance were *P<0.05, **P<0.01, ***P<0.001. Statistical analyses were carried out using GraphPad Prism or SPSS. Images were processed using Fiji ImageJ49 and analysed by manual counting with experimenter blinded to image identity throughout. Flow cytometry data were analysed and populations quantified using FlowJo V10.

Data availability

The data that support the findings of this study are available from the corresponding author on request.