Supporting Online Material. 3. Analysis of in vivo TLR4-mediated response in TRIF-deficient mice

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1 Supporting Online Material 1. Materials and Methods 2. Generation of TRIF-deficient mice 3. Analysis of in vivo TLR4-mediated response in TRIF-deficient mice 4. Measurement of LPS-induced IRAK-1 kinase activity in TRIF-deficient macrophages 5. Supporting online figures 6. Supporting online references 1. Materials and Methods Generation of TRIF-deficient mice The Trif gene was isolated from genomic DNA extracted from (ES) cells (E14.1) by PCR using TaKaRa LA Taq TM (TaKaRa). The targeting vector was constructed by replacing a 2.1-kb fragment encoding the entire TRIF ORF with a neomycin-resistance gene cassette (neo), and a herpes simplex virus thymidine kinase driven by PGK promoter was inserted into the genomic fragment for negative selection (Fig. S1a). After the targeting vector was transfected into ES cells, G418 and gancyclovir doubly resistant colonies were selected and screened by PCR and Southern blotting. omologous recombinants were micro-injected into C57BL/6 female mice, and heterozygous F1 progenies were intercrossed in order to obtain TRIF-deficient mice. TRIF-deficient mice and their wild-type littermates from these intercrosses were used for experiments. Reagents R-848 was kindly provided by the Pharmaceuticals and Biotechnology Laboratory, Japan Energy Corporation (S1). CpG oligodeoxynucleotides were prepared as described

2 previously (S2). LPS from Salmonella minnesota Re 595 prepared by phenolchloroform-petroleum ether extraction procedure, PGN from S. aureus and poly (I:C) were purchased from Sigma,Fluka and Amersham, respectively. Anti-phospho-JNK antibody was purchased from Cell Signaling. Anti-JNK1 and anti-erk1/2 antibodies were from Santa Cruz. Polyclonal anti-trif antibody was raised against corresponding to amino acids or of mouse TRIF for immunoprecipitation or immunoblotting, respectively. Polyclonal anti-irf-3 antibody was raised against corresponding to amino acids of mouse IRF-3. Anti-IgM antibody analysis was purchased from Jackson ImmunoResearch Laboratory. FITC-labeled streptavidin, PEconjugated anti-b220, biotin-conjugated anti-cd69, anti-cd86, and anti-i-a b antibodies were from Pharmingen. Electrophoretic Mobility Shift Assay Embryonic fibroblasts and lung fibroblasts (1x10 6 ) were stimulated with 10 µg/ml LPS, 50 µg/ml poly(i:c), and 10 ng/ml TNF-α for the indicated periods. Nuclear extracts were purified from cells and incubated with a specific probe for NF-κB DNA binding site, electrophoresed, and visualized by autoradiography as described previously (S3). Measurement of Proinflammatory Cytokine Concentrations Thioglycollate-elicited peritoneal macrophages were cultured in 96-well plates (5x10 4 cells per well) with the indicated concentrations of PGN, LPS, R-848, or CpG DNA. Concentrations of TNF-α, IL-6, IL-12 p40 in the culture supernatant was measured by ELISA according to manufacturer s instructions (Genzyme). B cell Proliferation Aassay Splenocytes (1x10 5 ) were cultured in 96-well plates for 24 hr with the indicated concentrations of poly(i:c), LPS, R-848 or CpG DNA. One microcurie of [ 3 ]- thymidine was pulsed for the last 12 hr and then [ 3 ] uptake was measured in a β-

3 scintillation counter (Packard). Flow Cytometric Analysis Two million splenocytes were cultured with 50 µg/ml poly(i:c), 10 µg/ml LPS, or 10 µg/ml anti-igm antibody. After 36 hr of culture, cells were harvested and stained with PE-conjugated anti-b220 antibody and biotin-conjugated anti-cd69, CD86 or I-A b antibody followed by streptavidin-fitc. Stained cells were analyzed on FACS Calibur using CellQuest software (Becton Dickinson). Western Blot Analysis and in vitro Kinase Assay Embryonic fibroblasts (1x10 6 ) were stimulated with 10 µg/ml LPS for the indicated periods. The cells were then lysed in a lysis buffer containing 1.0% Nonidet-P 40, 150 mm NaCl, 20 mm Tris-Cl (p 7.5), 1 mm EDTA and protease inhibitor cocktail (Roche). The cell lysates were dissolved by SDS-PAGE and transferred onto a PVDF membrane (BioRad). The membrane was blotted with anti-phospho-jnk or anti-jnk1 antibody, and visualized with an enhanced chemiluminescence system (NEN Life Science Product). IRAK-1 activity in cell lysates was measured by in vitro kinase assay as described (S4). Preparation of Lung Fibroblasts Lungs from mice were excised, washed in PBS, cut into small pieces, agitated, and digested enzymatically for 30 min at 37 C. The digestion buffer (10 ml/lung) was composed of 0.25% trypsin solution containing 400nM EDTA. Ice-cold complete DMEM medium was added to the resulting cell suspension. After centrifugation (1100 rpm, 5 min), pellets were resuspended in complete medium, then cultured on dish. Lung fibroblasts were used for each experiment in ten days after excision. Native PAGE Assay

4 Lung fibroblasts (1x10 6 ) and peritoneal macrophages (5x10 6 ) were stimulated with 50 µg/ml poly (I:C) and 1 µg/ml LPS, respectively, for the indicated periods, and then lysed. The cell lysates in native-page sample buffer (62.5mM Tris-Cl, p6.8, 15% glycerol and 1% deoxycholate) were separated on a native-page, and then immunoblotted with anti-irf-3 antibody as described previously (S4). 2. Generation of TRIF-deficient mice The mouse Trif gene consists of one exon. We constructed a targeting vector so that an entire exon was replaced with the neomycin resistance (neo) gene (Fig. S1a). Correctly targeted embryonic stem (ES) cell clones were used to generate mice with the mutated allele (Fig. S1b). Northern blot analysis indicated that peritoneal macrophages from the mutant mice did not express TRIF mrna (Fig. S1c). Immunoblot analysis of lung fibroblasts confirmed that the disruption of the Trif gene abolished the expression of TRIF protein (Fig. S1d). 3. Analysis of in vivo TLR4-mediated response in TRIF-deficient mice We investigated whether signaling through TRIF is required for the in vivo inflammatory response to LPS. All wild-type mice died within 24 h after a lethal dose of LPS injection, however TRIF-/- mice were significantly resistant to LPS-induced shock (Fig. S2a). Moreover, TRIF-/- mice showed profoundly defective levels of TNF-α and IL-6 production compared with wild-type mice (Fig. S2b,c). 4. Measurement of LPS-induced IRAK-1 kinase activity in TRIF-deficient macrophages Stimulation with LPS induced autophophorylation of IRAK-1 in wild-type macrophages,

5 but not in MyD88-deficient cells (S5). These results demonstrate that LPS-induced IRAK-1 activity is completely dependent on MyD88. In TRIF-/- macrophages, IRAK-1 was activated normally, indicating that MyD88-dependent TLR4 signaling pathway was intact (Fig. S3). 5 Supporting Online Figures Figure S1 Targeted disruption of the murine Trif gene. Figure S2 Measurement of LPS-induced shock in TRIF-deficient mice. Figure S3 LPS-stimulated autophosphorylation of IRAK-1 in TRIF-deficient cells.

6 Fig.S1_Yamamoto et al. S1a 1kb probe wild-type allele targeting vector neo 3.8kb SV-TK mutated allele neo 6.1kb ;incii S1b S1c S1d +/+ -/- +/- WT KO WT KO 6.1 kb TRIF β-actin TRIF ERK1/2 3.8 kb Figure S1 Targeted disruption of the murine Trif gene. a, The structure of the Trif gene, the targeting vector and the predicted disrupted gene. Closed box denotes the coding exon. Restriction enzymes:, incii. b, Southern blot analysis of offspring from the heterozygote intercrosses. Genomic DNA was extracted from mouse tails, digested with incii, electrophoresed and hybridized with the radiolabelled probe indicated in (A). Southern blotting gave a single 3.8-kb band for wild-type (+/+), a 6.1-kb band for homozygous (-/-) and both bands for heterozygous mice (+/-). c, Northern blot analysis of embryonic fibroblasts. Total RNA (10 µg) extracted from embryonic fibroblasts was electrophoresed, transferred to a nylon membrane, and hybridized using the TRIF fragment as a probe. The same membrane was re-hybridized with a β-actin probe. d, Endogenous expression of TRIF. Cell lysates prepared from embryonic fibroblasts were immunoprecipitated with anti-trif antibody. Then, the immunoprecipitants were immunoblotted with another anti-trif antibody. The same lysates were also blotted antibody against ERK1/2 to monitor protein expression.

7 Fig.S2_Yamamoto et al. survival rate (%) S2a S2b S2c (day) +/+ -/- (ng/ml) TNF- α +/+ -/ (hour) (ng/ml) IL (hour) Figure. S2 Measurement of LPS-induced shock in TRIF-deficient mice. a, Age-matched wild-type mice (n=5) and TRIF-/- mice (n=5) were intraperitoneally injected with 1.0mg LPS (E.coli O55:B5) and 20 mg D-GalN. Mortality was assessed daily for 4 days. b,c, Sera were taken after the indicated period of time and serum levels of (b)tnf-α and (c)il-6 were determined with specific ELISA assay. The data are means±s.d. of sera samples from 5 mice for each group at 1.5, 3, and 6 h, respectively.

8 Fig.S3_Yamamoto et al. LPS wild TRIF-/ min Auto IRAK-1 Figure S3 LPS-stimulated autophosphorylation of IRAK-1 in TRIFdeficient cells. Lysates from macrophages stimulated with LPS were immunoprecipitated with an antibody against IRAK-1. The kinase activity of IRAK-1 was measured by in vitro kinase assay (top). The same lysates were blotted with an antibody against IRAK-1 (bottom). Auto indicate auto-phophorylation.

9 6. Supporting online references S1.. emmi.et al. Nat. Immunol. 3, 196 (2002). S2.. emmi. et al. Nature 408, 740 (2000) S3. O. Adachi. et al. Immunity 9, 143 (1998). S4. S. Sato et al. Int. Immunol. 14, 783 (2002) S5. T. Kawai, O. Adachi, T. Ogawa, K. Takeda, S. Akira, Immunity 11, 115 (1999).