Ethics assertion
All animal experiments have been carried out in compliance with the Laws for the Care and Use of Laboratory Animals issued by the Ministry of Science and Know-how of the Folks’s Republic of China, which enforces the moral use of animals. The protocol was accredited by Institutional Animal Care and Use Committee (IACUC) at Institut Pasteur of Shanghai, Chinese language Academy of Sciences (Allow Quantity: P2019014).
Virus
Influenza virus pressure A/Puerto Rico/8/1934 (H1N1) was inoculated into the chorioallantoic fluid of 9-day-old specific-pathogen-free (SPF) rooster embryos, as beforehand described36. The titer comparable to the median deadly dose (LD50) was decided in grownup mice by intranasal inoculation. All viral an infection experiments have been carried out within the biosafety degree 2 laboratory at Institut Pasteur of Shanghai following the usual working protocols accredited by the institutional biosafety committee.
Mice
All mice used on this examine have been feminine mice on C57BL/6 background (6–9 weeks previous). WT (wild-type), Ifnar1−/− and Mavs−/− mice have been initially from the Jax lab, Irf3−/− mice have been initially from Dr. T. Taniguchi and shared by Dr. F. Shao37, Gpr43−/− mice have been bought from Cyagen Biotechnology Co., Ltd (https://www.cyagen.com/cn/zh-cn/sperm-bank-live/233079) and Nlrp3−/− mice had been described earlier than38,39. The mice have been routinely maintained in a pathogen-free animal facility at a temperature of 21 °C, relative humidity of fifty%-70%, and underneath a relentless 12 h gentle/darkish cycle, and got free entry to a daily chow food regimen (Cat# P1101F-25, Shanghai SLACOM) and water all through the examine at Institut Pasteur of Shanghai. Cohousing (Co-) of WT and Nlrp3−/− mice have been carried out at 1:1 ratio since weaning (3 weeks previous) until maturity (9 weeks previous), or the mice have been separated upon weaning in keeping with genotype, denoted as singly housed (Single-). All procedures have been carried out in compliance with a protocol accredited by the IACUC at Institut Pasteur of Shanghai, Chinese language Academy of Sciences, China.
Bone marrow-derived macrophages (BMDM)
BMDMs have been differentiated in keeping with the strategy described beforehand40. Briefly, bone marrow cells have been remoted by flushing femurs and tibia of 6- to 8-week-old WT, Nlrp3−/−, Irf3−/−, Gpr43−/− or Mavs−/− mice with DMEM medium (Hyclone, Cat# SH30809.01). Crimson blood cells have been lysed with purple blood cell lysis buffer (Beyotime), and bone marrow cells have been cultured in DMEM medium supplemented with 30% mouse fibroblasts L929 (ATCC CCL-1) conditioned medium, 10% FBS (AusGeneX, Cat# SA500S), 100 U/mL Penicillin and 100 μg/mL Streptomycin (Gibco), in addition to 50 μM β-Me (SIGMA). At day 3, recent DMEM medium and L929 cell (ATCC CCL-1) conditioned medium was added. BMDMs have been used on day 5 for siRNA transfection by electroporation or on day 6 for sodium acetate remedy plus polyI:C stimulation.
Virus an infection of mice
Mice have been anesthetized with Avertin (2,2,2-tribromoethanol, SIGMA) and contaminated intranasally with A/Puerto Rico/8/1934 (H1N1) in 25 uL of PBS (1 LD50 for 9-week-old WT mice). Mice have been both weighed and monitored each day or euthanized at numerous intervals for sampling. Mice have been humanely euthanized when an infection had progressed to a human endpoint at which the mice began to undergo. The contaminated mice of greater than 25% weight reduction have been handled as dying. Weight reduction and survival knowledge have been analyzed utilizing the GraphPad Prism v8.0 software program until acknowledged in any other case.
Depletion of commensal micro organism
Intestine microbiota elimination was carried out as beforehand described41. Briefly, mice have been handled with a cocktail of antibiotics containing ampicillin (1 g/L), metronidazole (1 g/L), neomycin (1 g/L), and vancomycin (0.5 g/L) in ingesting water for six weeks. Stool pellets (~0.05 g/mice) have been collected in 1 mL of Ringer’s de resolution (for 1 L preparation, NaCl 9 g, KCl 0.4 g, CaCl2·2H2O 0.25 g, and L-Cystcine·HCl 0.5 g, 121 °C autoclaved for 15 min) and homogenized. Completely different dilutions of the suspensions have been plated on mGAM (HB8518) and incubated at 37 °C both in an anaerobic incubator (80% N2:10% CO2:10% H2) or regular cardio situation for 48 h. Bacterial counts have been decided by colony-forming assay and the depletion effectivity was >99.9%. All of the antibiotics used right here have been bought from Sangon Biotech (Shanghai) Co.
Intestine microbiota profiling
The bacterial DNA extraction from fecal samples of mice have been carried out as beforehand described42. A sequencing library of the V3–V4 areas of the 16S rRNA gene was constructed following the producer’s directions (Half #876 15044223Rev.B; Illumina Inc., San Diego, CA, USA) with enchancment as beforehand described43, and sequenced on the Illumina MiSeq platform (Illumina, Inc., San Diego, CA, USA). The uncooked paired-end reads have been processed and analyzed utilizing the QIIME2 v2018.11. Demultiplexed sequence knowledge was imported into QIIME2 v2018.11, adapters and primers have been trimmed. ASVs from every pattern have been inferred by utilizing the DADA2 pipeline for filtering, dereplication, pattern inference, merging of paired-end reads and chimera identification. Within the strategy of operating the DADA2 pipeline, primarily based on the standard profile of the information, ahead and reverse reads have been trimmed accordingly to make sure that the median high quality rating for every place is above 32. The taxonomy of all ASVs have been annotated by SILVA (v132) reference database44. All samples have been rarefied to 10,000 per pattern for downstream evaluation. Principal coordinate evaluation (PCoA) of ASVs primarily based on Bray–Curtis distance was carried out utilizing QIIME2 v2018.11. The amplicon sequence variants (ASVs) that have been considerably altered between two teams have been recognized utilizing the algorithm LDA Impact Dimension (LEfSe)20.
1H NMR (nuclear magnetic resonance) spectroscopy
Detection of metabolites was carried out by Shanghai Metabolome Institute (SMI)-Wuhan. Fecal samples have been ready by dissolving 50 mg of feces in 600 μL of 0.1 M deuterated Na+/Okay+ buffer (for 0.1 L preparation, 1.844 g Okay2HPO4·3H2O, 0.315 g NaH2PO4·2H2O, 0.001 g TSP (sodium 3-(trimethylsilyl) propionate-2,2,3,3-d4), 0.01 g NaN3, PH7.5) and fast frozen-thawed thrice with liquid nitrogen. The combination was homogenized with a tissue lyser (90 s, 20 Hz) and centrifuged at 13,200 × g for 10 min at 4 °C. The supernatant (550 μL) was transferred to a 5 mm NMR tube. The fecal NMR spectra have been acquired at 298 Okay underneath an automated pattern changer system (BACS, Bruker Biospin, Germany) utilizing a Bruker AVIII 600 MHz spectrometer outfitted with an inverse cryoprobe (working at 600.13 MHz for 1H). One dimensional 1H NMR spectra have been acquired for every fecal pattern utilizing NOESYGPPR1D sequence (RD-90°-t1−90°-tm−90°-ACQ). Water suppression was achieved with irradiation on the water peak through the recycle delay (RD) of two s and a mixing time ™ of 100 ms. t1 was set to 4 μs. The 90° pulse size was adjusted to 11 μs, and 32 transients have been collected into 32k knowledge factors for every spectrum with a spectral width of 20 ppm. Knowledge evaluation had been described earlier than45.
Sequence-guided micro organism isolation
Recent fecal samples from Nlrp3−/− mice have been collected and blended in anaerobic sterile Ringer’s de resolution proven in Depletion of Commensal Micro organism part above in an anaerobic workstation (Don Whitley scientific Ltd, 798 Shipley, UK). The diluted suspension with Ringer’s de resolution was plated onto mGAM and incubated underneath anaerobic situation (80% N2, 10% CO2, and 10% H2) at 37 °C for 4 days. The 16S rRNA gene V3 area of every colony was obtained utilizing the primer pairs listed in Supplementary Desk 1. The obtained 16S rRNA gene V3 sequences have been subjected to Denaturing Gradient Gel Electrophoresis (DGGE) and aligned with the ASV1 or ASV4 enriched within the intestine of singly housed Nlrp3−/− mice. ASV4-associated pressure was efficiently remoted utilizing a beforehand reported mGAM medium (Nissui 05426) and named B. pseudolongum NjM1. The 16S rRNA sequence of B. pseudolongum NjM1 was aligned in GenBank and the 16S rRNA gene sequences of consultant strains within the genus Bifidobacterium have been chosen to construct a phylogenetic tree by utilizing the Neighbor-Becoming a member of technique in MEGA 6.
Entire-genome sequencing and evaluation of B. pseudolongum NjM1
Entire-genome DNA of B. pseudolongum NjM1 was extracted with QIAamp BiOstic Bacteremia DNA Package (QIAGEN) and sequenced with the PromethION platform (Nextomics Biosciences, Wuhan, China). Subreads of the sequence have been assembled right into a single full chromosome utilizing the HGAP 2.3.0 pipeline46. Protein-coding sequences (CDSs), tRNAs, and rRNAs have been predicted and annotated utilizing the Prokka 1.12 pipeline47. Practical annotation of genes was carried out utilizing the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.
Quick-chain fatty acid (SCFA) profiling for B. pseudolongum NjM1
SCFA concentrations in B. pseudolongum NjM1 tradition supernatant have been decided utilizing gasoline chromatography/mass spectrometry (GC/MS) as beforehand described48. Briefly, B. pseudolongum NjM1 was cultured in mGAM medium underneath anaerobic situation (80% N2, 10% CO2, and 10% H2) at 37 °C for 8 h, and centrifuged at 16,000 × g for 15 min at 4 °C. B. pseudolongum NjM1 tradition supernatant was filtered by a 0.22 μm nylon filter (EMD Millipore). An aliquot (50 μL) of the supernatant was acidified by including 25 μL 50% (v/v) sulfuric acid and vortexing for 10–15 s. The natural acids have been extracted by including 100 μL of diethyl ether and vortexing for 10–15 s and standing for two min. After centrifugation at 12,000 × g for five min at 4 °C, the supernatant containing SCFAs was measured by GC on an Agilent 6890 (Agilent Applied sciences, CA, USA) outfitted with flame ionization, thermal conductivity detectors, capillary columns and GC ChemStation software program.
B. pseudolongum NjM1 inoculation in mice
B. pseudolongum NjM1 was cultured in mGAM medium underneath anaerobic situation (80% N2, 10% CO2, and 10% H2) at 37 °C for 8 h. The harvested bacterial cells have been washed twice with pure mGAM and resuspended in mGAM, and added with equal quantity of fifty% glycerol to a density of seven.4 × 1010 cells/mL and saved at −80 °C till 10-fold dilution and inoculation by gavage with 1.48 × 109 cells for every mouse. The tradition supernatant of B. pseudolongum NjM1 was used to deal with mice by intranasal administration.
Mice feeding with HAMS or HAMSA diets and acetate measurements
Diets HAMS (high-amylose maize starch) (fiber (4.8% cellulose and 15% high-amylose resistant starch), 18.1% protein, 7.1% fats and 17.7 mj/kg digestible power) and HAMSA (acetylated HAMS) (fiber (4.8% cellulose and 15% acetylated high-amylose resistant starch), 18.1% protein, 7.1% fats and 17.7 mj/kg digestible power) have been produced by XieTong Biology, NanJing, China. Mice have been fed with the diets HAMS or HAMSA for five weeks (beginning at 3 weeks of age as beforehand described21, diets have been refreshed thrice per week) earlier than subjected to influenza A viral an infection experiments. Cecal, fecal samples and serum have been processed for SCFA evaluation utilizing gasoline chromatography on the finish of the viral an infection.
Pulmonary histopathology
For histopathologic examination, lungs have been collected from mice on the indicated days, fastened by immersion in 10% buffered formalin for at the very least one week at 4 °C, processed, embedded in paraffin, sectioned, after which stained with hematoxylin and eosin (H&E). The peribronchial irritation was scored in keeping with Barends et al49., ranked as absent (0), minimal (1), slight (2), average (3), marked (4), or extreme (5). Slide evaluation was carried out in a blinded method.
Plaque assay of IAV
Lungs have been weighed and homogenized in 1 mL of PBS, bronchoalveolar lavage fluid (BALF) was harvested in 1 mL of PBS, and the supernatants of PR8-infected BMDMs have been harvested. They have been centrifuged at 400 × g for five min at 4 °C. The supernatants containing influenza virus have been diluted serially with 0.2% BSA (bovine serum albumin) in PBS. MDCK cells (ATCC CCL-34) have been grown in 48-well cell tradition plates to supply a confluent monolayer, after which washed with PBS, contaminated with serially diluted supernatants, and incubated at 37 °C for 1 h for virus absorption. Unabsorbed virus particles have been washed away with PBS, after which 0.5 mL of 1×DMEM (SIGMA, Cat# D5796) medium containing 0.2% BSA, 100 U/mL Penicillin and 100 μg/mL Streptomycin, 1.2% colloidal microcrystalline cellulose (Avicel®) and a couple of μg/mL TPCK-treated trypsin, was added to every nicely. After incubation for 48 h at 37 °C in 5% CO2 incubator, the MDCK cells have been fastened with 4% paraformaldehyde (PFA), after which incubated with rabbit anti-influenza A Nucleoprotein/NP antibody (SinoBiological, Cat# 11675-T62, dilution 1:2000) adopted with goat-anti-rabbit HRP. Coloration improvement was carried out with True BlueTM Peroxidase Substrate (KPL), plaques in every nicely have been counted, after which the outcomes have been averaged and multiplied by the dilution issue for calculation of viral titers.
Quantitative real-time PCR
Complete RNA was extracted from homogenized lung or BMDMs with TRIzol reagent (SIGMA) in keeping with the producer’s directions. Synthesis of cDNA was carried out with a GoScriptTM Reverse Transcription equipment (Promega). Actual time quantitative PCR was carried out with the SYBR Inexperienced qPCR Grasp Combine (TOYOBO) on an ABI 7900 HT Quick Actual-Time cycler (Utilized Biosystems). The expression of goal genes was normalized to expression of housekeeping gene Gapdh (glyceraldehyde-3-phosphate dehydrogenase). The qPCR primers used on this examine have been listed in Supplementary Desk 1.
Macrophage depletion
WT mice have been pretreated with sodium acetate (SA) or ingesting water after which intranasally administered with 100 μL of clodronate (LIPOSOMA, C28J0620) to deplete macrophages within the lung. The cell variety of alveolar macrophages in BALF have been analyzed by move cytometry.
BALF associated experiments
The bronchoalveolar lavage fluid (BALF) was harvested in 1 mL of PBS on day 3 post-infection and centrifuged at 400 × g for five min at 4 °C. The supernatants have been collected and frozen at −80 °C for enzyme-linked immunosorbent assay (ELISA) and virus titration. The purple blood cells have been lysed, and remaining cells have been counted with a hemocytometer and stained with fluorochrome-conjugated antibodies for move cytometry.
Movement cytometry
Cells from BALF or BMDMs have been washed with FACS buffer (1 × PBS containing 0.5% BSA and a couple of mM EDTA) after which blocked with FcR blocking antibody (anti-Mouse CD16/CD32 (clone 2.4G2, BD Pharmingen, Cat# 553142, dilution 1:100)) earlier than floor staining with BV605 anti-Mouse CD45.2 (clone 104, BD Horizon, Cat# 563051, dilution 1:160), APC-Cy7 anti-Mouse CD11b (clone M1/70, BD Pharmingen, Cat# 561039, dilution 1:160), PE-Cy7 anti-Mouse F4/80 (clone BM8, Biolegend, Cat# 123114, dilution 1:160) and AF488 anti-GPR43 (Bioss, Cat# bs-13536R-A488, dilution 1:160). For intracellular Ca2+ detection, BMDMs have been handled with 250 μM sodium acetate (SA) (S5636, SIGMA) for twenty-four h, washed with PBS thrice, after which incubated with 2 μM of Fluo-4 AM (S1060, Beyotime) for 45 min at 24 °C. Lifeless cells have been excluded utilizing 7AAD (BD Pharmingen, Cat# 559925, dilution 1:500) and knowledge have been acquired utilizing a BD Fortessa move cytometer with BD FACSDivaTM software program v6 after which analyzed with FlowJo_v10 software program.
Enzyme-linked Immunosorbent Assay (ELISA)
Mouse IFN-α and IFN-β protein ranges have been measured with mouse IFN-α ELISA equipment (BMS6027, Invitrogen) and mouse IFN-β ELISA equipment (42400-1, PBL Assay Science) in keeping with the producers’ directions.
Immunoblotting
The cells have been incubated in lysis buffer containing 50 mM Tris-HCl (PH 7.5), 150 mM NaCl, 1% NP40 (v/v), 1.0% SDS (m/v), 1 mM NaF, 1 mM Na3VO4 and protease inhibitor cocktail at 4 °C for 30 min on a shaker, after which the cell lysates have been blended with SDS loading buffer. The protein-loading buffer combination was boiled to denature at 100 °C for 10 min earlier than the SDS-PAGE gel electrophoresis for protein separation. After electrophoresis, the proteins have been wet-transferred to nitrocellulose membranes (Millipore), blocked with 5% BSA in 1×TBS containing 0.05% Tween-20 after which blotted for indicated proteins utilizing the antibodies listed in Supplementary Desk 2. The uncropped blot photographs could be present in Supply Knowledge file.
siRNA design and transfection
SiRNAs (small interfering RNAs) concentrating on Gpr43 or Luciferase GL2 have been ordered from NIBS, Beijing. Oligonucleotides for siRNAs concentrating on Gpr43 or Luciferase GL2 have been listed in Supplementary Desk 1. siRNAs have been transfected to BMDMs by electroporation (100 pmol of siRNAs per 1 × 106 BMDMs) with the electroporation resolution and cuvettes (Mirus Ingenio®) and a nucleofector system (Lonza) outfitted with a program O-013 for 48 h earlier than sodium acetate remedy.
Confocal fluorescence microscopy
BMDMs have been handled with or with out 250 μM sodium acetate for twenty-four h after which transfected with 200 ng polyI:C utilizing transfection reagent Lipofectamine 2000 for 1 h. The cells have been fastened with 4% paraformaldehyde (PFA) for 15 min, blocked with 1 × PBS containing 5% goat sera and 0.3% Triton X-100 for 1 h after which sequentially incubated with rabbit anti-MAVS (Cat# 4983, rodent particular, CST) for 12 h at 4 °C, goat-anti-rabbit IgG (AF555 conjugated) for two h at room temperature, rabbit anti-GPR43 (AF488 conjugated) (bs-13536R-A488, Bioss) for two h at room temperature and DAPI for 1 min at room temperature. The fluorescence photographs have been collected on a laser seize confocal microscope (Olympus FV1200) utilizing separate laser excitation to keep away from any cross-interference between completely different fluorophores. For NLRP3 colocalization with MAVS or GPR43, Nlrp3−/− BMDMs have been contaminated with PRP-eGFP-mNlrp3 retrovirus and pretreated with or with out 250 μM sodium acetate for twenty-four h after which transfected with 200 ng polyI:C for 1 h. The cells have been fastened, blocked, after which incubated with rabbit anti-MAVS (Cat# 4983, rodent particular, CST) or rabbit anti-GPR43 (bs-13536R, Bioss) for 12 h at 4 °C, goat-anti-rabbit IgG (AF555 conjugated) for two h at room temperature and DAPI for 1 min at room temperature. The fluorescence photographs have been collected.
Co-immunoprecipitation assay
HEK293T cells (ATCC CRL-3216) have been transfected with the plasmids utilizing transfection reagent Lipofectamine 2000 (invitrogen). At 36 h post-transfection, the cells have been lysed with lysis buffer (50 mM Tris-HCl PH7.5, 150 mM NaCl, 1% NP40 (v/v), 0.5% SDS (m/v)) supplemented with a proteinase inhibitor cocktail (Roche), 1 mM NaF and 1 mM Na3VO4 for 30 min at 4 °C. The cell lysates have been centrifuged at 1000 × g for 10 min at 4 °C and 10% supernatants have been used as enter, and 90% supernatants have been 10-fold diluted with 0.5% NP40 (50 mM Tris-HCl PH7.5, 150 mM NaCl, 0.5% NP40 (v/v)) after which incubated with both 1 μg mouse anti-FLAG (clone M2, SIGMA, Cat# F3165, dilution 1:1000) or 1 μg mouse anti-V5 (clone AMC0506, ABcolonal, Cat# AE017, dilution 1:200) or 1 μg mouse anti-GFP (clone 5G4, CST, Cat# 55494, dilution 1:50) on a curler (QILINBEIER-206) in a single day at 4 °C. The Protein A/G Magnetic Beads for IP (bimake) have been added to the supernatants and incubated for 1 h on the curler at 4 °C, washed thrice with 0.5% NP40, after which boiled in 2×Western loading buffer for five min at 95 °C for immunoblotting.
Semidenaturing detergent agarose gel electrophoresis for MAVS aggregation
Semidenaturing detergent agarose gel electrophoresis (SDD-AGE) was carried out for the detection of MAVS aggregation in keeping with a broadcast protocol with minor modifications25. In short, BMDMs have been handled with or with out 250 μM sodium acetate for twenty-four h after which transfected with 200 ng polyI:C utilizing transfection reagent Lipofectamine 2000 for 1 h. Crude mitochondria have been extracted utilizing a mitochondria isolation equipment (Cat# 89874, Thermo ScientificTM) in keeping with the producer’s directions on Reagent-based Technique after which resuspended in 1×pattern buffer (0.5 × TBE, 10% glycerol, 2% SDS, 0.0025% bromophenol blue) and loaded onto a vertical 1.5% agarose gel (BIOWEST). After electrophoresis within the operating buffer (1×TBE and 0.1% SDS) for 35 min with a relentless voltage of 100 V at 4 °C, the proteins have been transferred to a PVDF membrane (Millipore) for immunoblotting.
Ca2 + chelator experiment and inhibitor experiment
BMDMs have been pretreated with BAPTA-AM (Cat# HY-100545, MCE), BAY 11-7085 (Cat# HY-13453, MCE), SB203580 (Cat# 5633, CST), PD98059 (Cat# 9900, CST), or 25 μM SP600125 (Cat# 8177, CST) for 1 h and 250 μM sodium acetate (S5636, SIGMA) for twenty-four h after which transfected with 200 ng polyI:C utilizing Lipofectamine 2000. Relative Nlrp3 and Ifna1 expression to Gapdh was decided by quantitative real-time PCR.
Statistical evaluation
GraphPad Prism v8.0 software program (La Jolla, CA, USA) and IBM SPSS Statistics 23 have been used for knowledge evaluation. Statistically vital distinction was decided by two-tailed Scholar’s t check for 2 teams or one-way ANOVA with Tukey’s post-hoc check or Dunnett’s post-hoc check for 3 or extra teams. Survival curves have been in contrast with the Log-rank (Mantel–Cox) check. p < 0.05 was thought-about a statistically vital.
Reporting abstract
Additional info on analysis design is accessible within the Nature Portfolio Reporting Summary linked to this text.
