Reagents

Ultrapure LPS-B5, ATP, nigericin, Pam3CSK4, poly(I:C), CL264 and Hygromycin B Gold were obtained from InvivoGen. P. alecto ASC-specific monoclonal antibody (mouse IgG2b) was generated by GenScript’s monoclonal antibody service. Rabbit polyclonal anti-ASC (AL177) (human/mouse) was purchased from Adipogen. Goat polyclonal anti-dog IL-1β (ab193852) (cross-reactive to P. alecto), rabbit polyclonal anti-mouse IL-1β (ab9722) and monoclonal antibody (mAb) to IAV nucleoprotein (ab20343) were from Abcam. mAb to β-actin (A2228) was from Sigma-Aldrich and mAb to GFP and variants (including mCitrine) were from Roche (11814460001). Anti-dsRNA mAb J2 was purchased from SCICONS and anti-mouse/rabbit/goat/monkey horseradish peroxidase (HRP)-conjugated secondary antibodies were from Santa Cruz. mPlum antibody from Origene was used to stain mPlum. Human mAb to MERS-CoV spike glycoprotein (m336) was provided by D. S. Dimitrov (NIH)58. Alexa 488/568/647 Zenon labelling kits (Invitrogen) were used for direct labelling of antibodies for confocal microscopy and ImageStream. Protein G agarose beads (Millipore) were used to purify P. alecto IL-1β-Fc cloned and expressed from HEK293T cells. Polyclonal macaque serum (#5244 D42, infected by PRV3M, produced in-house) was used for immunoblot analysis.

Viruses

Human H1N1 IAV strain A/NWS/33 (ATCC # VR-219) was propagated in Vero, clone E6 cells in DMEM, 0.3% BSA, 25 mM HEPES and 1 µg ml−1 TPCK-treated trypsin. P. orthoreovirus virus, PRV3M (Melaka virus) and MERS-CoV were propagated in Vero B4 cells in DMEM, 2% FBS. At a virus-induced cytopathatic effect of 80–90%, viruses were harvested, clarified by centrifugation, and the virus-containing supernatant was stored at −80 °C. PRV3M was further purified using ultracentrifugation and resuspended in DPBS. To titrate IAV, A/NWS/33 Madin Darby canine kidney (MDCK, ATCC #CCL-34) cells were infected with IAV for 1 h at 37 °C. After 1 h incubation, the medium was replaced with plaque medium (DMEM, 0.8% Avicel, 0.3% BSA, 25 mM HEPES and 1 µg ml−1 TPCK-treated trypsin). Plaques were fixed with 4% paraformaldehyde at 48 h post-infection and stained with 0.5% crystal violet, and titres were expressed in p.f.u. ml−1. PRV3M and MERS-CoV titres were determined by limiting dilution. In brief, tenfold serial diluted virus was added into a 96-well plate containing 1 × 104 Vero B4 cells per well. Cells were observed for cytopathatic effect and the titres were expressed as TCID 50 ml−1. All work with live MERS-CoV was performed in BSL3 containment.

Plasmids

Expression constructs for ASC–mPlum, NLRP3–mCitrine, IL-1β-HA and empty vectors were generated in the retroviral backbone of pQCXIH (Clontech). NLRP3 chimaeras between human NLRP3 and the P. alecto NLRP3 isoform 1 were generated by overlap extension PCR. ASC-3×Myc and 3×Myc construct were generated in pDual GC (Agilent Technologies). P. alecto IL-1β-Fc construct containing the IL-2 signal sequence was generated from pFUSE-hIgG1-Fc plasmid (Invivogen). pVSV-G envelope vector for retroviral packaging was obtained from Clontech. pCI-Caspase-1 construct encoding human pro-caspase-1 and pCMV-pro-IL-1β construct encoding mouse pro-IL-1β were from Addgene. P. alecto and M. davidii genes were cloned from Omniscript (Qiagen)-generated cDNA from bat tissues. All constructs were prepared with endotoxin-free plasmid maxi-prep kits (Omega Bio-tek).

Cells

All procedures in this study dealing with animal samples are in compliance with all relevant ethical regulations. Specifically, capturing and processing of bats (P. alecto) in Australia was approved by the Queensland Animal Science Precinct & University of Queensland Animal Ethics Committee (AEC#SVS/073/16/USGMS) and the Australian Animal Health Laboratory (AAHL) Animal Ethics Committee (AEC#1389 and AEC#1557). Where possible, wild bats with irreparable physical damage (torn wings) already scheduled for euthanasia were utilized. Processing of bats has been described previously59. Human blood was obtained from healthy donors with consent, approved by the National University of Singapore Institutional Review Board (NUS-IRB reference code H-18–029). PBMCs were isolated by Ficoll-Paque Plus (GE Healthcare) density gradient centrifugation from P. alecto or human blood samples and stored in liquid nitrogen. Wild-type C57BL/6 mice were obtained with permission from the Singhealth IACUC committee. P. alecto and mouse splenocytes were isolated by grinding the spleen through a 100 μm cell strainer followed by red blood cell lysis. P. alecto bone marrow cells were processed and differentiated with recombinant P. alecto macrophage colony-stimulating factor (M-CSF) over 6 days into BMDMs or over 8 days with recombinant P. alecto granulocyte-macrophage colony-stimulating factor (GM-CSF) into BMDCs, as described previously49. Mouse bone marrow cells were harvested and differentiated into BMDMs or BMDCs over 6 days or 8 days of cultures using 10 ng ml−1 mouse M-CSF or 20 ng ml−1 mouse GM-CSF recombinant proteins (R&D System)60. Immortalized P. alecto and M. davidii kidney cells have been described previously59,61. GP2–293 retroviral packaging cells were obtained from Clontech. The NLRP3-knockout iMACs have been described previously62 and were provided by E. Latz (University of Bonn). Frozen PBMCs, splenocytes and bone marrow cells were thawed in 37 °C for 2 min, washed twice with warmed medium and recovered at 37 °C for 2 h in RPMI 1640 medium containing 10% (vol/vol) FBS before experiments. PBMCs, splenocytes, BMDMs and reconstituted iMACs were cultured in RPMI 1640 medium containing 10% FBS (Biological Industries). MDCK, Vero, GP2–293, HEK293T, PaKi and MdKi cells were cultured in DMEM (Gibco) medium supplemented with 10% FBS. Sodium pyruvate and NEAA (Life Technologies) were supplemented into the culture media of GP2–293 cells during retroviral packaging.

Generation of HEK293T, PaKi and MdKi reporter cells

GP2–293 cells, stably expressing gag and pol proteins, were co-transfected with pVSV-G envelope vector and human or bat ASC–mPlum, or mPlum-only empty vector for 48 h. Retroviral supernatants were collected, centrifuged at 500g for 10 min and filtered through a 0.45 μm hydrophilic polyethersulfone filter (Millipore). HEK293T, PaKi or MdKi cells were transduced with a low MOI of retrovirus for 24 h to obtain a single viral copy per transduced cell, followed by Hygromycin B antibiotic selection 72 h post transduction for at least 1 month to ensure stable expression. HEK293T stable cells were further (fluorescence-activated cell sorting (FACS)-sorted for low mPlum-positive cells of similar fluorescent intensity to ensure no spontaneous activation of ASC–mPlum.

Reconstitution of NLPR3 in macrophages

Retroviral supernatants for human or bat NLRP3–mCitrine, or mCitrine-only, were similarly generated to transduce mouse NLRP3-knockout iMACs by first centrifuging cells with the retroviral supernatants at 300g for 1 h at 37 °C, then incubating for 24 h. At 72 h after the transduction, cells were FACS-sorted for low mCitrine-positive cells of similar fluorescent intensity.

Inflammasome activation assays

PBMCs, BMDMs, BMDCs, splenocytes or reconstituted iMACs were seeded at 1 × 106 ml−1 into 96-well, 24-well or 12-well plates. Cells were primed with 10 ng ml−1 LPS for 3 h. The medium was removed and replaced with serum-free RPMI 1640 medium or medium containing 5 mM ATP or 6.7 μM nigericin for 30 min for detection of ASC specks or 60 min for LDH release assay and IL-1β detection by immunoblots, ELISA or targeted mass spectrometry. For ImageStream detection of ASC speck formation, HEK293T, PaKi and MdKi reporter cells were seeded overnight into 24-well plates and transfected with 100 ng (HEK293T) or 300 ng (PaKi or MdKi) per 24-well plate of the indicated NLRP3–mCitrine or mCitrine-only empty vectors using Fugene 6 (Promega) for HEK293T and Lipofectamine 3000 (Thermo) for Paki or Mdki, according to the manufacturer’s instructions. At 24 h (HEK293T) or 48 h (PaKi or MdKi) after transfection, cells were harvested by trypsinization and resuspended in FACS buffer for analysis. For immunoblotting, inflammasome system was reconstituted in HEK293T reporter cells by transfecting 50 ng indicated NLRP3–mCitrine, 10 ng human pro-caspase-1 and 190 ng mouse pro-IL-1β constructs per 24-well plate. Both supernatants and cells were collected for immunoblot analysis.

Viral infection of cells

For IAV and PRV3M infections, BMDMs, BMDCs or reconstituted iMACs were first primed with 10 ng ml−1 LPS for 3 h, washed and then infected with IAV (MOI = 0.5, 1, 2, 5 or 10) for 2 h or PRV3M (MOI = 1, 2, 5 or 10) for 4 h. Subsequently, the medium was replaced with AIM V serum-free medium (Invitrogen). At 24 h post infection, cells were collected for detection of ASC specks and immunoblots, and the cell-free supernatant for IL-1β immunoblots or ELISA and virus titrations. For MERS-CoV virus infections, PBMCs were first primed with 10 ng ml−1 LPS for 3 h, washed and then infected with a series of MOIs (0.25, 0.5 or 1) for 1 h. Lower MOIs of MERS-CoV were able to elicit a robust activation of the inflammasome in human PBMCs and hence were used in this study. The medium was replaced with AIM V serum-free medium (Invitrogen) for an additional 23 h before harvesting the cells for detection of ASC specks post-fixation and the cell-free supernatant for IL-1β ELISA and virus titration. Inhibitor MCC950 (50 μM; Selleckchem) was added 1 h before the virus infection in human PBMCs.

Confocal microscopy

Human or bat PBMCs were seeded into 24-well plates with coverslips (#1.5 thickness) inside the wells. After treatment, the cells were fixed with 4% paraformaldehyde with 0.37% gluteraldehyde in Tris-buffered saline (TBS) at room temperature for 20 min. Cells were then washed twice with TBS, permeabilized with 0.1% Triton X-100 for 10 min, blocked with 5% BSA in TBS for 30 min, stained with primary antibody (anti-ASC) pre-labelled with Zenon labelling kits in TBS for 1 h and washed three times. Nuclei were stained with DAPI. Coverslips were subsequently mounted onto glass slides with Mowiol 4.88. Z-stack confocal images were acquired on a Leica TCS SP8 machine with a ×100 objective, followed by quantification of ASC specks with Imaris 9.2.0 software for at least 10 fields per sample. Representative images were processed by maximum intensity projection of Z-stack with Image J 2.0.0.

ImageStream imaging flow cytometry

Cell supernatants were transferred to a 96-well V-bottom plate and centrifuged at 500g for 5 min at 4 °C. Cells were incubated with ice-cold 5 mM EDTA in PBS for 10 min, followed by pipetting to detach cells. Detached cells were combined with cell pellets from the supernatants, centrifuged again, and resuspended with 4% paraformaldehyde in TBS. Fixed cells were permeabilized with 0.3% Triton X-100 + 2% FBS + 1% NGS (normal goat serum) for 15 min at 4 °C, washed twice in wash buffer (TBS + 2% FBS), stained with pre-labelled primary antibodies for 1 h in TBS + 2% FBS + 1% NGS, washed twice, then resuspended in FACS buffer (PBS + 2 mM EDTA + 1% FBS). Nuclei were stained with DAPI. Cells were acquired using INSPIRE software on an Amnis ImageStreamX Mk II imaging flow cytometer using ×40 magnification. At least 10,000 single cells were acquired per sample and analysed with the inbuilt IDEAS software. First, cells in focus were gated using the bright-field gradient r.m.s. values. Single cells with an intermediate bright-field area and a high aspect ratio were selected. For PBMCs, the monocyte population was gated and separated from the lymphocyte population using bright-field area and side scatter. Based on the DAPI intensity, sub-G 0 /G 1 cells were excluded. Double-positive cells of similar fluorescent intensity for ASC–mPlum and NLRP3–mCitrine or virus-infected cells with positive staining for IAV nucleoprotein, dsRNA or MERS-CoV spike protein were also gated. ASC speck formation was then analysed by gating on a small area and a high maximum pixel value of ASC signal. Mean pixel intensity, size and circularity score (average distance of the object boundary from its centre divided by the variation of this distance) were measured for detected ASC specks.

LDH release assay

Cytosolic LDH is rapidly released into the supernatant medium following disruption of the plasma membrane. Cell culture media were collected and centrifuged at 500g for 5 min at 4 °C. The release of LDH was measured in the cell-free supernatants using a Cytotoxicity Detection Kit PLUS (LDH) from Roche. The kit was used according to the manufacturer’s instructions. The percentage of LDH release was calculated as LDH release (%) = (experiment value – low control)/(high control – low control) × 100. Low control is the baseline LDH release from untreated cells, while high control is the maximum LDH release from cells treated with lysis solution.

Immunoblot analysis

Cells were lysed in lysis buffer63 containing cOmplete ULTRA protease inhibitor cocktail and PhosSTOP phosphatase inhibitors (Roche). Cell-free supernatants were directly used or concentrated by methanol–chloroform precipitation and dissolved in SDS–sample buffers. Proteins were separated by 6–15% SDS–PAGE gels, transferred onto 0.45 μm polyvinylidene difluoride membrane with a Trans-Blot Turbo transfer system (Biorad). Membranes were blocked with 5% BSA in TBS-T (0.1% Tween-20) for 1 h and incubated with specific primary antibodies overnight in a cold room in 5% BSA in TBS-T. Membranes were washed and incubated with HRP-conjugated secondary antibody for 1 h. Membranes were developed using Amersham ECL Prime Western blotting detection reagent (GE Healthcare) and signals were detected with a myECL Imager (Thermo Scientific). Anti-GFP antibody was used to detect NLRP3–mCitrine by immunoblots.

ELISAs

ELISAs for human/mouse IL-1β in cell-free supernatants were performed according to the manufacturer’s instructions (R&D Systems; DY201 and DY401). Sandwich ELISA for bat IL-1β was generated using the goat anti-dog IL-1β (ab193852) as the capturing antibody and rabbit anti-mouse IL-1β (ab9722) as the detection antibody. Bicarbonate/carbonate coating buffer (50 mM), OptEIA assay diluent (BD bioscience), donkey anti-rabbit HRP-conjugated secondary antibody (Santa Cruz), 3,3′,5,5′-tetramethylbenzidine (TMB) chromogen solution (Invitrogen) and TMB stop solution (VWR) were used in the assay. Purified P. alecto IL-1β-Fc recombinant proteins were used as standards.

TLR stimulations and qPCR

BMDMs, BMDCs or PBMC of bat and mouse/human were seeded at 1 × 106 ml−1 into a 96-well plate. Cells were stimulated by various concentrations of TLR ligands for 3 h: LPS-B5 (10, 100 and 1,000 ng ml−1), Pam3CSK4 (10, 100 and 1,000 ng ml−1), CL264 (0.1, 1 and 10 μg ml−1) and poly(I:C) (0.1, 1 and 10 μg ml−1). Harvested tissues of mice or bats were homogenized using silicon-carbide sharp particles (BioSpec Products) in the FastPrep-24 5G Homogenizer (MP Biomedicals). Tissue and cellular RNA was extracted using an RNeasy Mini kit and an RNeasy Plus Micro Kit (Qiagen), respectively. RNA was converted into cDNA using a QuantiTect reverse transcription kit (Qiagen). qPCR reactions were prepared using the SensiFAST SYBR No-ROX Kit (Bioline) and were run on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using the following cycling condition: 95 °C for 5 min, 40 cycles of 95 °C for 10 s and 55 °C for 30 s, with a melt curve cycle. Targeted genes (NLRP3, ASC, caspase-1 and IL-1β) were normalized to the geometric mean of three housekeeping genes (GAPDH, β-actin and SNRDP3) in P. alecto or mouse tissues after correction for PCR efficiency by standard curves from sample serial dilution. Fold induction of cytokines (NLRP3, IL-1β, IL-6 and TNF-α) was normalized to housekeeping (GAPDH) and compared to mock treated. The relative abundance of exon 7-positive and exon 7-negative NLRP3 isoforms was determined by quantifying ‘total’ NLRP3 and exon 7-positive isoforms using specific primers targeting a common region and exon 7, respectively. Serially diluted plasmids of both isoforms were used to perform standard curve analysis. qPCR primers for target and reference genes are listed in Supplementary Table 4.

Isoform analysis

Illumina HiSeq RNA–seq sequencing was performed on both CD11b+MHC-II− (putative monocytes/macrophages) and CD11b+MHC-II+ (putative DCs) of P. alecto splenocytes (n = 5) as described previously49. We used TopHat to map the pooled reads from all data sets to the genomic locus of P. alecto NLRP3 and used Cufflinks64 and Velvet65 to perform de novo assembly. TopHat was then used to map the pooled reads to the two splice variants identified. For PCR validation of isoforms, RNA extraction from the spleen and thymus of P. alecto or the spleen and kidney from M. davidii was performed using the RNeasy mini kit (Qiagen) and cDNA was produced using an Omniscript (Qiagen) reverse transcription kit. PCR amplification was performed using Q5 polymerase (NEB).

Targeted mass spectrometry analysis

Cells and concentrated cell-free supernatants (methanol–chloroform precipitation) were lysed and denatured in 8 M urea/50 mM Tris-HCl buffer pH 8.0. Proteins were reduced with 25 mM TCEP for 20 min at 25 °C and alkylated with 55 mM 2-chloroacetamide (CAA) for 30 min, in the dark, at room temperature. Before digestion, samples were diluted with 100 mM triethylamonium bicarbonate buffer. Protease digestion was carried out with LysC enzyme (Wako) for 4 h, followed by trypsin (Promega) treatment for 18 h at 25 °C (1:100, enzyme:protein ratio). On the following day, samples were acidified with 1% trifluoroacetic acid and peptides were purified by Sep-Pak C18 cartridges (Waters). Elution of peptides was performed with 0.5% acetic acid, 80% acetonitrile followed by peptide concentration using a vacuum concentrator system. Vacuum dried peptides were subsequently analysed on an EASY-nLC 1000 (Thermo) chromatography system coupled with Orbitrap Fusion mass spectrometer (Thermo). Each sample was separated on 70 min gradient (0.1% formic acid in water and 99.9% acetonitrile with 0.1% formic acid) using a 50 cm × 75 μm inner diameter EASY-Spray Reverse Phase Column (C-18, 2 μm particles, Thermo). Data were acquired in targeted PRM mode together with IL-1β peptide internal standards and peptide mass inclusion list. For acquisition, an Orbitrap analyser with ion targets and resolution (OT-MS 4e5 ions 120k; PRM tMS/MS 3e5 ions 15k) was used. The total area MS1 of each peptide was analysed and exported using Skyline software (version 3.7.0)66. Values for endogenous peptides were normalized by those for the heavy isotope-labelled internal standards.

Evolutionary analysis of mammalian NLRP3

The coding sequences for NLRP3 were collected form GenBank and Ensembl for 27 species representing the major groups of mammals (Supplementary Tables 2 and 3). After manual correction, the multiple sequence alignments for the full-length gene and its three domains (PYD, NACHT and LRR) were separately subjected to evolutionary analysis. The branch model from PAML was used to test whether the bat ancestor lineage is under positive selection and statistically different to the rest of the phylogeny, using a likelihood ratio test67.

Statistical analysis

Data are presented as mean and s.d. or s.e.m. of multiple biological replicates or independent experiments (as indicated). Statistical analysis was performed using GraphPad Prism software. Results were tested for significance using unpaired two-tailed Student’s t-tests when two conditions were compared. One-way or two-way ANOVA involving one or two independent variables and Bonferroni’s multiple comparisons test were performed if multiple samples or conditions were compared. Data were considered significant if *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Exact P values are provided in Supplementary Table 5.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.