Cells and virus
THP-1 cells were cultured in RPMI-1640 medium (Thermo-Fisher Scientific, MA, USA) containing 10% fetal bovine serum (FBS) (GE, Boston, USA). Human rhabdomyosarcoma (RD) cells were obtained from Dr. Shih Shin-Ru and grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% FBS, 1% non-essential amino acids, 1% L-glutamine, and 1% penicillin/streptomycin (all from Thermo-Fisher Scientific, MA, USA). NLRP3-KD THP-1 cells (were gifted by Dr. Jan Martel, Chang Gung University, Immunology Center) are THP-1 cells that infected with lentiviral vectors that carried NLRP3 shRNA. All cells were incubated in a 37 oC incubator that equilibrated with 5% CO2. The EV-A71 strain 2231 (TW/2231/98), strain 70811 (TW/70811/08) was isolated from the Clinical Virology Laboratory of Chang Gung Memorial Hospital (Linkou, Taiwan), as well as EV-A71 strain BrCr (USA/BrCr/1970), was amplified using RD cells, and the titer was quantified by plaque forming assays.
Isolation of PBMCs and differentiation of PBMCs toward macrophages
Human peripheral blood samples were collected after approval by IRB (Chang Gung Medical Foundation institutional review board, IRB2019050059). All the research process was performed in accordance with IRB guidelines and regulations. The informed consent was obtained from all subjects and/or their legal guardians. Mononuclear cells were harvested by Ficoll-Paque method. Briefly, peripheral blood was mixed with PBS (1:1). The diluted cell suspension was then layered on Ficoll-Paque (GE Healthcare Life Sciences, MA, USA) (volume 2:1) in a 50 mL canonical tube. After centrifugation at 400 x g for 20 minutes, the top layer was aspirated. The mononuclear cell layer in the interface was then transferred to a new tube. To differentiate the harvested PBMCs toward macrophages, RPMI medium supplemented with 20 ng/mL M-CSF (Peprotech, NJ, USA) and 1% human serum was added and cultured for 5 days.
Reagents
1 µg/mL LPS (Sigma-Alderich, MO, USA) was used to stimulate cells for activation. phorbol 12-myristate 13-acetate (PMA) was prepared at 200 µM in DMSO (Dimethyl sulfoxide). 200 nM PMA was used to stimulate THP-1 cells differentiation. Ac-YVAD-cmk (Sigma-Alderich, MO, USA) was prepared at 10 mM in DMSO. 1 µM Ac-YVAD-cmk was used to inhibit caspase-1 activation. Z-IETD-FMK (R&D systems, MN, USA) was prepared at 20 mM in DMSO. 20 µM Z-IETD-FMK was used to treat PMA primed THP-1 after virus infection.
Virus infection
Cells were seeded in culture plates and cultivated at different time points. The seeded cells were rinsed by PBS twice and serum-free medium was added with the virus at a specified multiplicity of infection (M.O.I.). Adsorption was performed at 37 oC for 1 hour, and the medium was then decanted. The cells were washed by PBS twice and 2% FBS containing medium was added for further incubation.
RNA extraction and RT-qPCR
Trizol reagent (Life Technologies, Gaithersburg, MD) was applied to extract total RNA from cell samples. The cells were lysis by Trizol reagent and then mixed with chloroform. After 5 min, the homogenate was centrifugated at 12,000 x g for 15 min at 4 oC. The aqueous phase was transferred to a new tube and added with an equal volume of isopropanol and then incubated for 10 min. The mixture was centrifuged at 12,000 x g for 8 min at 4 oC. After the centrifugation, the supernatant was removed. 500 µL 7 % ethanol was used to wash the RNA pellet then centrifugated at 12,000 x g for 5 min at 4 oC. Discard the supernatant and then air dried the RNA pellet. The RNA pellet was dissolved by sterile water and quantified the concertation of RNA by NonoDrop technology (Thermo-Fisher Scientific, MA, USA). 1 µg of total RNA was used to synthesize cDNA. RevertAid First Strand cDNA Synthesis Kit (Thermo-Fisher Scientific, MA, USA) was ap-plied according to the manufacturer's instructions. To detect the expression of target genes, specific primers for TNF-α, IL-6, IL-1β, EV-A71 5’UTR were used (Table 1). qPCR assays were carried out on 384-well plates and analyzed by a Roche Lightcycler 480 instrument (Roche, Basel, SW). Triplicate for each sample in qPCR analysis and 18s rRNA was used as a reference gene. The relative expression level of each gene was analyzed by 2−ΔΔCT method.
Table 1
qPCR primer used in this study.
Gene | | sequence |
Human 18s rRNA | Forward | 5’- GTA ACC CGT TGA ACC CCA TT -3’ |
Reverse | 5’- CCA TCC AAT CGG TAG TAG CG -3’ |
EV-A71 5’UTR | Forward | 5’- CCC TGA ATG CGG CTA ATC C -3’ |
Reverse | 5’- ATT GTC ACC ATA AGC AGC CA -3’ |
Human TNF-α | Forward | 5’- CAT GAT CCG GGA CGT GGA GC -3’ |
Reverse | 5’- CTG ATT AGA GAG AGG TCC CTG -3’ |
Human IL-6 | Forward | 5’- GGA GAC TTG CCT GGT GAA AA -3’ |
Reverse | 5’- GTC AGG GGT GGT TAT TGC AT -3’ |
Human IL-1β | Forward | 5’- ACA GAT GAA GTG CTC CTT CCA -3’ |
Reverse | 5’- GTC GGA GAT TCG TAG CTG GAT -3’ |
Protein isolation and Western blot
The cultured cells were harvested at indicated time point. After washed by PBS twice, the cell lysates were collected using protein lysis buffer (1% NP-40, 50 mM Tris, and 150 mM NaCl) containing protease inhibitors cocktail (Bioshop, Ontario, Canada). After incubated on ice for 30 min, centrifugation was performed at 13,000 rpm for 10 min at 4 oC. The Supernatants were harvested, and protein concentrations were measured by Protein Assay Dye Reagent (Bio-Rad Laboratories, CA, USA). Proteins (30 µg) were separated by 1 % or 1 % SDS-polyacrylamide gel electrophoresis, and then transferred onto a poly-vinylidene fluoride membrane (PVDF) (GE, MA, USA). The membrane was then blocked with 5% skim milk in Tris-buffered saline Tween-20 buffer (TBST) (20 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, and 0. % Tween 20) at room temperature for one hour. After washed by TBST for twice, the membrane was then incubated with anti-EV-A71 3D Ab (1:10000, was gifted by Professor Shih Shin-Ru), anti-EV-A71 VP0 (1:1000, Millipore, MA, USA), anti-IL1β Ab (1:1000, Cell Signaling Technology, MA, USA), anti-NLRP3 Ab (1:1000, Cell Signaling Technology, MA, USA), anti-caspase-1 Ab (1:1000, Santa Cruz Biotechnology, TX, USA), anti-caspase-8 Ab (1:1000, Cell Signaling Technology, MA, USA), Anti-RIG-I Ab (1:1000, Enzo Life Sciences, NY, USA), anti-TLR3 Ab (1:1000, Abcam, CAMB, UK), anti-p-P65 Ab (1:1000, Cell Signaling Technology, MA, USA), anti-lamin Ab (1:1000, Santa Cruz Biotechnology, TX, USA). The unbound antibodies were then removed by washing with TBST three times. Horseradish peroxidase conjugated secondary antibodies (1:5000, Jackson ImmunoResearch Laboratories, Pennsylvania, USA) were incubated for 1 hour. Finally, the target proteins were visualized with a Western Lightning Chemiluminescence reagent (PerkinElmer, MA, USA) and detected by ChemiDoc imaging system (Bio-Rad Laboratories, CA, USA).
siRNA transfection
The siRNAs targeting for NLRP3, caspase-1, RIG-I, and TLR3, as well as scramble siRNA, were used in this study (all from Sigma-Aldrich, MO, USA). The siRNA was prepared in 100 µM with RNase-free distilled water. 100 nM siRNA was used to transfect into the cells with Lipofectamine RNAiMAX (Thermo-Fisher Scientific, MA, USA) in opti-MEM medium (Thermo-Fisher Scientific, MA, USA) for 48 hr. After transfection, the cells were infected with EV-A71 and harvested the sample at various hours post-infection.
plaque assay
The total virus was harvested at different time points after virus infection. RD cells were seeded on 6-well plate at a density of 5 x 105 cell per well. After incubation, the collected virus was serially diluted by 0% FBS DMEM medium. RD cells were washed by PBS twice then infected by diluted virus medium. After absorption, the medium was removed and replaced by 2% FBS DMEM medium containing 0.3% agarose. After 96 hours of incubation, the medium was removed and the cells were stained by crystal violet.
ELISA
The culture medium was collected at different time points. Centrifugation was performed at 5,000 rpm for 10 min at 4 oC then the supernatants were harvested. IL-1β ELISA kit was obtained from Invitrogen (Thermo-Fisher Scientific, MA, USA). The an-ti-IL-1β antibody was coating on the 96 well plate overnight at 4 oC. After incubation, 96-well plate was washed by washing buffer once. Blocking the 96 well plate by assay buffer for 1 hr. Aspirate wells and then 100 µL standards and samples were added into each well with detection antibody. The 96-well plate was incubated at 700 rpm for 2 hours at room temperature. Aspirate wells and then wash by wash buffer for five times. The working streptavidin-HRP was added into each well and then incubated at 700 rpm for 30 min at room temperature. The plate was washed for five times. TMB (3, 3', 5, 5'-tetramethylbenzidine) solution was added into each well. Stop the reaction using 2 M H2SO4 solution. Detect the absorbance at 450 nm and 650 nm by microplate reader (BioTek, VT, USA).