Materials and reagents. Ac-YVAD-CMK (caspase-1 inhibitor) and LPS derived from Pseudomonas aeruginosa were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nigericin and flagellin derived from Pseudomonas aeruginosa were purchased from InvivoGen (San Diego, CA, USA). Primary antibodies: anti-β-catenin (WB) and anti-β-actin from Sigma-Aldrich; anti-TREM2(WB, IP) from Abcam (Cambridge, MA, USA); anti-β-catenin (IP), anti-phosphorylated β-catenin (S33/37, S552, S675), anti-phosphorylated Akt and anti-phosphorylated GSK-3β from Cell Signaling Test (CST; Beverly, MA, USA); anti-Caspase-1 (p10), anti-Lamin B from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-Caspase-1 (p20), anti-NLRP3, anti-NLRC4 and anti-ASC from Adipogen (San Diego, CA, USA). Detail catalog information of antibodies and reagents provide in Supplementary Table 1.
Bacterial strains. Pseudomonas aeruginosa strain (19660), Staphylococcus aureus strain (25923), Streptococcus pneumoniae strain (49136), Salmonella Typhimurium (13311) and Escherichia coli strain (25922) was purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Salmonella Typhimurium were grown on LB plates medium at 37°C. Streptococcus pneumoniae were grown at Columbia blood plate medium at 37°C in a humidified incubator with 5% CO2.
Experimental animals. Six-week-old C57BL/6 (B6) mice were purchased from Sun Yat-sen University Animal Supply Center. TREM2-/- (KO) mice in B6 background were generously provided by Marco Colonna (Washington University School of Medicine) and bred in Sun Yat-sen University Animal Supply Center. Caspase-1-/-(Casp1 KO) mice in B6 background were purchased from Jackson Laboratory (Bar Harbor, ME, USA). All animals were housed in Specified Pathogen Free (SPF) environment. All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of Sun Yat-sen University (Guangzhou, Guangdong, China).
Isolation and culture of bone marrow-derived macrophages. Mouse bone marrow-derived macrophages (BMDMs) were isolated and differentiated as reported previously. In brief, WT, TREM2 KO or Casp1 KO mice were sacrificed by Spinal dislocation and the bone marrow in femurs was flushed out and cultured in DMEM containing 10% FBS, 1% penicillin-streptomycin, 1% L-glutamine (all from Invitrogen, Carlsbad, CA, USA) and 30% (vol/vol) conditioned medium of L-929 cells (mouse fibroblast cell line), as a source of M-CSF. BMDMs were obtained as a homogeneous population of adherent cells after 7 days culture. The BMDM purity was routinely > 95% as assessed by flow cytometry.
Cell culture and transient transfection. iBMDMs were kindly provided by Feng Shao (National Institute of Biological Sciences, Beijing). Murine macrophage-like RAW264.7 cells (TIB-71) were obtained from ATCC. These cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% L-glutamine. Cells were incubated at 37°C in a humidified incubator with 5% CO2. According to the manual of Lipofectamine™ 2000 (Invitrogen), iBMDMs or RAW264.7 were transiently transfected with pcDNA3.1 vector or plasmids encoding wild-type (WT) or mutant (S675A, S552A) β-catenin, or specific small interfering RNA against TREM2 (siTREM2), β-catenin (siβ-cat) or GSDMD (siGSDMD) vs scrambled control siRNA (siNC). Each siRNA was mixed by two siRNAs that target different sequences of the same specific gene. All siRNAs were synthesized by Ruibo Biotechnology (Guangzhou, Guangdong, China). Flagellin transfection was performed according to the manual of DOTAP Liposomal Transfect Reagent (Sigma-Aldrich).
Lentivirus transduction and establishment of iBMDM cells with stable β-catenin expression. A lentiviral plasmid encoding β-catenin with 45’ serine, 41’ threonine, 37’ serine, and 33’ serine mutation was purchased from Addgene (Seattle, WA, USA). Production of lentivirus vectors was carried out as described previously [27]. Briefly, HEK293T cells (ATCC, Manassas, VA, USA) were transfected with 2.5 µg lentiviral plasmid, 0.8 µg VSV-G enveloping plasmid and 1.7 µg pCMVDR8.2 packaging plasmid. After 48 hours post-transfection, cell supernatants were collected and centrifuged at 4000 g for 5 min to discard cell fragments. Viral titer was measured by the HIV p24 Antigen ELISA kit (Zeptometrix, Buffalo, NY). Next, iBMDM cells were infected by β-catenin-lentivirus or vector-lentivirus at a multiple of infection (MOI) of 10 for 6 hours in the presence of 8 mg/ml polybrene (Sigma). Infected cells were sorted by limiting dilution assay till single cell clones of β-catenin-iBMDM (β-cat-iBM) or control vector-iBMDM (vec-iBM) cells that expressing mCherry fluorescence protein were observed by fluorescence microscopy (Olympus IX53, Olympus, Japan) and then collected for further culture and assays.
Lactate dehydrogenase (LDH) cytotoxicity assay and ELISA. iBMDMs or BMDMs were treated as indicated above, cell supernatants and lysate were collected to analyze cell death by LDH cytotoxicity assay, using CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, WI, USA). ELISA was conducted to analyze IL-1β secretion in cell supernatants, using specific ELISA kits from BD Biosciences. Each sample was assayed in duplicate. Results are shown as the means ± SEM of three independent experiments.
Real-time PCR. Total RNA was isolated from cell pellets using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction, and quantitated using a NanoDrop 2000C Spectrophotometers (Thermo Scientific, West Palm Beach, FL, USA). 1 µg of total RNA was reverse-transcribed to produce cDNA by using RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). Then the cDNA was amplified using SYBR green master mix (TaKaRa, Mountain View, CA, USA) following the manufacturer’s protocol. Quantitative Real-time PCR reactions were performed using a CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). Relative mRNA levels were calculated after normalization to the level of β-actin.
Western-blot. Cells were washed three times with ice-cold PBS and then lysed in lysis buffer containing 1 mM phenylmethylsulfonyl fluoride, 1% (v/v) protease inhibitor cocktail, 1% (v/v) phosphorylase inhibitor cocktail, and 1 mM dithiothreitol (all from Sigma, St. Louis, MO, USA). Nuclear and cytoplasmic protein was isolated by using a Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, West Palm Beach, FL, USA). Cell supernatants were mixed with methanol and chloroform and centrifuged in 4°C to precipitate the protein, and then protein pellets were dissolved in lysis buffer. Equal amounts of cell lysates were resolved by SDS-PAGE and then transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% Bovine Serum Albumin PBST (pH 7.4 PBS, 0.5% Tween20) at room temperature for 1 hour and incubated at 4°C overnight with the respective primary antibodies, followed by a second incubation at room temperature for 1 hour with appropriate HRP-conjugated secondary antibodies. Finally, blots on the membranes were visualized with Plus-ECL (KeyGEN, Nanjing, China) according to the manufacturer’s protocol.
ASC oligomerization assay. Cells were lysed in 300 µl Triton Buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, and EDTA-free protease inhibitor cocktail]. 30µl of the lysates used as input control. The rest of cell lysates were centrifuged at 6,000 × g at 4°C for 15 min and the pellets were washed twice and resuspended with Triton Buffer, followed by cross-linking with 2 mM disuccinimidyl suberate (DSS) for 30 min at room temperature. Then the pellets were collected by centrifugation, and dissolved in SDS loading buffer. Precipitated pellets and soluble lysates were simultaneously immunoblotted using anti-ASC Ab.
Co-immunoprecipitation assay. BMDMs from B6 mice or iBMDM cell were challenged with LPS (3 hours) or LPS plus ATP for 1 hours, and then cells were collected and lysed with Pierce™IP lysis buffer (Thermo Fisher Scientific). Subsequently, cell lysates were incubated with Antibody of ASC or NLRP3, vs their respective isotype- matched IgG control, at 4°C overnight and then with Protein G agarose beads (Thermo Fisher Scientific) for additional 4 hours. The agarose beads were collected by centrifugation and then washed four times with IP washing buffer. Proteins were eluted in reducing sample buffer and analyzed by immunoblot.
Flow cytometry evaluation of cell death. PI staining kit (KeyGen Biotech, Nanjing, China) was used to stain cells according to the manufacturer's instructions. After bacteria challenge, cells were washed thrice with binding buffer, and then 5×105 cells were incubated with 500 µl binding buffer containing 5 µl PI for 5 minutes. Finally, cells were collected and analyzed using a Beckman Coulter EPICS XL/MCL flow cytometer (Beckman Coulter Inc, Fullerton, CA, USA).
Intracellular bacterial killing assay. Intracellular bacterial killing efficiency was assessed using plate count method as described previously [28]. Briefly, after indicated treatments, BMDMs or iBMDMs were stimulated with LPS (100ng/ml) for 12 hours, followed by Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae and Escherichia coli infection at an MOI of 20. After an hour infection, cells were washed thrice with cold PBS and cultured for 30 min in complete medium containing 200µg/ml gentamicin to remove extracellular bacteria. After that, cells in one of the duplicate wells were lysed for calculation of internalized bacteria [CFU (1 h)]. Cells in the other duplicate well were incubated in complete medium for another one hour. After cell lysis and bacterial culture, the number of survived intracellular bacteria [CFU (2 h)] was calculated. Killing efficiency of the intracellular bacteria was calculated using the following equation: intracellular bacterial killing= [CFU (1 h) –CFU (2 h)] / CFU (1 h) × 100%.
Statistical analysis. The differences between two groups or among three or more groups were analyzed by using an unpaired two-tailed Student’s t test, or one-way analysis of variance (one-way ANOVA) with Bonferroni’s posttest, respectively. Differences were considered statistically significant when the P value was < 0.05.