Generation of VISA-Knockout mice
The VISA targeting vector was constructed by replacing a 4.3 kb genomic region of murine VISA gene, including exon2, 3, 4 and 5, with a PGK-Neo selection cassette (). The targeting construct was transfected into TC1 ES line (derived from 129Sv strain). G418 resistant ES clones were screened by Southern-blot analysis. Positive clone was microinjected into C57BL/6 blastocysts to produce chimeric mice. Chimeric mice were bred to C57BL/6 mice to obtain germline transmission. The heterozygous F1 progenies were intercrossed to obtain VISA−/− mice. The VISA−/− mice were then backcrossed to C57BL/6 strain for 4 generations. The experiments were done on VISA−/− and their WT littermates. The genotypes of the mice were determined by genomic PCR. TLR7−/− mice were kindly provided by Dr. Philippa Marrack (National Jewish Health). Animals were generated at the National Jewish Health Mouse Genetics Core Facility and used at 6–8 wks of age. Animal care and handling was performed as per IACUC Guidelines (protocol # AS-2519-04-12).
Recombinant mouse IFN-β protein (Abcam,ab84998), Loxoribine (Invivogen,tlrl-lox), R848 (Invivogen,tlrl-r848), CpG-ODN 1668 (Invivogen,tlrl-1668), anti-IRAK1 Ab (Cell Signaling, #4504), anti-IRAK4 Ab (Cell Signaling, # 4363), anti-MyD88 Ab (Santa Cruz, sc-11356), anti-TLR7 Ab (eBioscience,14-9079), anti-TLR9 Ab (eBioscience,14-9092), anti-TLR4 Ab (R&D system, MAB2759) and anti-TLR3 Ab (R&D system,MAB3005), anti-IκBα Ab (Cell Signaling, cat# 9242), anti-NF-κB 2 (p52) Ab (Santa Cruz, sc-298). Spleens from MAVS knockout mice were a gift from Dr. Zhijian Chen at UT Southwestern Medical School.
B cell purification and culture
Splenic B cells were purified from 6~8 weeks-old mice using Miltenyi Biotec B cell isolation kit as per the manufacturer’s instructions. Purified B cells were cultured at 5.0×106/ml in IMDM (HyClone, Logan, UT) supplemented with 5% FBS (Biosource, 200p-500HI), L-glutamine (2mM), penicillin (100 UI/ml), 100 μg/ml streptomycin, gentamycin (50 μg/ml), sodium pyruvate (1mM) and 2-ME (50 μM). Cells were cultured at 37°C with 7% CO2. Allculture reagents were from Life Technologies (Gaithersburg, MD).
Calcium mobilization ([Ca+2]i)
Intracellular calcium concentration was measured as previous described (20
). Briefly, cells were loaded with Indo-1AM (Molecular Probes, Eugene, OR) for 30min at 37°C, then washed and suspended in IMDM supplemented with 2% FCS at 106
cells/ml. Cells were stimulated with goat anti-mouse IgM-μF(ab′)2
(Jackson ImmunoResearch, 115-006-020). Data were collected on LSR II and analyzed by Flow-Jo software (Tree Star, Inc., San Carlos, CA).
Mouse IFN-α, TNF and IL-12 ELISA
IFN-α, TNF and IL-12 were measured in culture supernatants using commercial ELISA kits (IFN-α kit, PBL Biomedical Laboratories, VeriKine™ Mouse Interferon-Alpha ELISA Kit, 42100, TNF kit, eBioscience, 88-7324-22 and IL-12 p70 kit, R&D Systems, M1270)
Flow Cytometric assay of Immunofluorescence
Cells were stained with PerCp-anti-B220 (BD Pharmigen; RA3.3A1), FITC-anti-CD86 (BD Pharmigen, GL1) and APC-anit-CD69 (Biolegend, H1.2F3), APC or PE-anti- CD23(BD Pharmingen, clone B3B4), FITC-anti CD21(BD Pharmigen, 7G6), PE-anti-IgD(Southern Biotech, 11–26), PerCP-anti-IgM (Biolegend, R6-60.2). FACS was performed using a FACScan flow cytometer (BD Biosciences) and analyzed by FlowJo (CellQuest).
Immunoprecipitation and SDS-PAGE and Immunoblotting
Cells were lysed in cell lysis buffer (20mM Tris pH7.5, 150mM NaCl, 1mM EDTA, 1% Triton, 1mM PMSF, 10mM sodium pyrophosphate, 2mM Na3VO4, 10mM NaF, 1 μg/ml aprotinin, 1 μg/ml Leupetin) at 4°C for 1h. Cell lysates were centrifuged at 12,000g at 4°C for 10 min, Immunoprecipitation was done in the lysates with indicated Ab-conjugated sepharose beads. The immunoprecipitates were run on a SDS-PAGE and probed with indicated Abs.
Quantitative real-time PCR (qPCR)
qPCR were done as previously described (21
). Briefly, RNA was isolated using RNeasy kit (Qiagen, 74104) and cDNA was synthesized using the Superscript III First-Strand Synthesis kit (Invitrogen, 11752-050). qPCR reactions were done in an ABI PRISM 5700 Sequence Detection System (Applied Biosystems). Results were normalized to GAPDH control and represented as fold change over WT control. Primers used in the qPCR are
- TLR7-F: 5′-CCACAGGCTCACCCATACTTC-3′;
- TLR7-R: 5′-GGCATGTCCTAGGTGGTGACA-3′;
- TLR9-F: 5′-TGGGCCCATTGTGATGAAC-3′;
- TLR9-R: 5′-TTGGTCTGCACCTCCAACAGT-3′;
- IFNβ-F: 5′-CCCTATGGAGATGACGGAGA-3′;
- IFNβ-R: 5′-TCCCACGTCAATCTTTCCTC-3′;
- IFNα4-F: 5′-TCCATCAGCAGCTCAATGAC-3′;
- IFNα4-R: 5′-AGGAAGAGAGGGCTCTCCAG-3′;
- GAPDH-F: 5′-TCAACAGCAACTCCCACTCTTCCA-3′;
- GAPDH-R: 5′-ACCCTGTTGCTGTAGCCGTATTCA-3′.
B cells were swollen in 500 μl Buffer A (10 mM Hepes, 10 mM NaCl, 5 mM EDTA, 1.5 mM MgCl2, 10 μg/ml aprotinin and leupeptin, 2mM dithiothreitol, and 1mM phenylmethylsulfonyl fluoride, pH 7.8) at 4 °C for 10 min then lysed in equal volume of Buffer B (Buffer A plus 1.2% Nonidet P-40). Samples were vortexed for 10 s. The pellets were collected and washed with Buffer A, then suspended in 4.5 μl of Buffer C (Buffer A plus 10% glycerol). The suspension was mixed with 5 μl of 4.1 M NaCl and left at 4 °C for 30 min. The nuclear extract supernatant was collected and quantified with Bio-Rad protein assay kit. The NF-κB targeting oligonucleotide, GGGGACTTTCCC (Santa Cruz Biotechnology) labeled with [γ-32P]ATP by T4 DNA kinase, was added into 20 μg of nuclear extract and 0.8 μg of poly(dI-dC) in binding buffer (20 mM Tris, 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Nonidet P-40, 5% glycerol, pH 7.5) at room temperature for 15 min. The mixture was fractionated in 5% acrylamide gel prepared in 0.5× Tris borate-EDTA. The gel was dried and subjected to autoradiography.
3[H] uptake assay
B cells (3×105 cells/well in 150 μl medium in 96 well plates) were stimulated with indicated TLR agonists for 24 h, then pulsed for 8 h with (one microcurie of 3[H]thymidine/per well (Amersham Bioscience). Incorporation of 3[H] thymidine was quantified by a liquid scintillation beta counter (PerkinElmer).
Adoptive cell transfer
Purified splenic B cells were labeled with CFSE (2.5μM) for 3 min in PBS buffer. Cells were then washed with PBS containing 5% FBS followed by 3 more washes with PBS. The labeled cells (1 × 107) were transferred (i.v.) to recipient mice (WT or VISA−/−).