Journal: BMC Molecular Biology

Article Title: Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies

doi: 10.1186/1471-2199-9-3

Figure Lengend Snippet: ABI inventoried Taqman ® gene expression assays

Article Snippet: TLR8 , Hs00152972_m1.

Techniques: Gene Expression

Journal: Nature chemical biology

Article Title: Small-molecule inhibition of TLR8 through stabilization of its resting state

doi: 10.1038/nchembio.2518

Figure Lengend Snippet: (a) Chemical structures of CU-CPT8m and 6 (negative control), concentration-response curve and dose-dependent cytotoxicity of CU-CPT8m in HEK-Blue TLR8 cell line. Data was normalized to a DMSO control (data are mean ± SD ; n = 3 independent experiments). (b) ITC thermogram of CU-CPT8m titrated into hTLR8 to determine binding affinity and stoichiometry (representative of one independent experiment). The raw data are presented on top and the integrated peak areas are shown and fitted below. Mean K d = 0.22 μM; stoichiometric binding N = 0.5. (c) Specificity test for CU-CPT8m (1 μM) with TLR-specific agonists used to selectively activate different HEK-Blue TLR-overexpressing cells in the presence or absence of 1 μM CU-CPT8m (data are mean ± SD ; n = 3 independent experiments). (d) TNF-α and IL-8 mRNA level in R848 treated HEK-Blue TLR8 cells in the presence and absence of 1 μM CU-CPT8m or the negative control, 6 (10 μM). Data are the average quantification of two independent experiments. (e) Dose-dependent response of CU-CPT8m on TLR8-mediated TNF-α production in THP-1 cells with indicated concentration of CU-CPT8m or 6 . Data are mean ± SD ; n = 3 independent experiments. (f) Dose-dependent response of CU-CPT8m or 6 on TLR8-mediated TNF-α production in PBMC cells induced by 1 μg/mL R848. Data are mean ± SD ; n = 3 independent experiments.

Article Snippet: TLR8 antibody (Novus Biologicals; NBP2-24972) was added in PBS containing 1% FBS, then incubated for 10 min.

Techniques: Negative Control, Concentration Assay, Control, Binding Assay

Journal: Nature chemical biology

Article Title: Small-molecule inhibition of TLR8 through stabilization of its resting state

doi: 10.1038/nchembio.2518

Figure Lengend Snippet: (a) Front (top) and side (bottom) views of the unliganded (left, PDB ID 3W3G), TLR8/ CU-CPT8m (middle) and TLR8/R848 (right, PDB ID 3W3N) complexes. TLR8 and its dimerization partner TLR8* are colored green and cyan, respectively. The distances between the C-termini of the two protomers of TLR8 dimer (TLR8/ CU-CPT8m ) is similar to that of the unliganded dimer (right). Superimposition of the TLR8 structure complexed with CU-CPT8m onto the corresponding unliganded TLR8 segment (a.a. 32–816) produces root-mean-square deviation (RMSD) values of 2.4 Å. The ligand molecules are illustrated by space-filling representations. The C, O and N atoms of the ligands are colored yellow, red, and blue, respectively. (b) Close-up view of antagonist binding site of unliganded TLR8 (left) and TLR8/ CU-CPT8m (right). Water molecules are indicated by red filled circles. (c) Schematic representation of interactions between CU-CPT8m and the TLR8 protein. The hydrophobic pocket and hydrogen bonds are shown as dashed gray arcs and dashed red lines, respectively.

Article Snippet: TLR8 antibody (Novus Biologicals; NBP2-24972) was added in PBS containing 1% FBS, then incubated for 10 min.

Techniques: Binding Assay

Journal: Nature chemical biology

Article Title: Small-molecule inhibition of TLR8 through stabilization of its resting state

doi: 10.1038/nchembio.2518

Figure Lengend Snippet: LRR8, LRR11-13, LRR15-16, and LRR17-18 are colored yellow, green, blue, and purple, respectively. In the bottom panel, the antagonist and agonist are illustrated by yellow and orange circles. Interactions between ligands and protruding loop regions are shown by dashed arrows. TLR8 utilized LRR11-13 in common for both agonist and antagonist binding on one side of the interface, while on the other side LRR17*-18* and LRR15*-16* for agonist and antagonist binding, respectively. Binding of agonist (e.g. R848 ) brings two TLR8 C-termini to a closer distance to initiate downstream signaling; while binding of antagonists (e.g. CU-CPT8m , CU-CPT9b ) at the antagonist binding site stabilizes inactive TLR8 dimer with C-termini further apart, preventing TLR8 from activation.

Article Snippet: TLR8 antibody (Novus Biologicals; NBP2-24972) was added in PBS containing 1% FBS, then incubated for 10 min.

Techniques: Binding Assay, Activation Assay

Journal: Nature chemical biology

Article Title: Small-molecule inhibition of TLR8 through stabilization of its resting state

doi: 10.1038/nchembio.2518

Figure Lengend Snippet: (a) Chemical structure of CU-CPT9a and CU-CPT9b. (b) Close-up view of antagonist binding site (left) and its schematic representation of TLR8/ CU-CPT9b (right). The C, O and N atoms of the ligands are colored yellow, red, and blue, respectively. Water molecules mediating the ligand recognition are indicated by red filled circles and hydrogen bonds by dashed lines. (c) Dose-dependent dimerization of TLR8. Elution profiles of gel filtration chromatography of TLR8 with CU-CPT9b (left) and R848 (right) at various concentrations. Retention volume and normalized absorbance at 280 nm (A 280 ) are shown on the left, and retention volume of TLR8 peak is plotted against its molar ratio (ligand/TLR8) on the right (representative of one independent experiment).

Article Snippet: TLR8 antibody (Novus Biologicals; NBP2-24972) was added in PBS containing 1% FBS, then incubated for 10 min.

Techniques: Binding Assay, Filtration, Chromatography

Journal: The Journal of Clinical Investigation

Article Title: p53-responsive TLR8 SNP enhances human innate immune response to respiratory syncytial virus

doi: 10.1172/JCI128626

Figure Lengend Snippet: (A) Graphical location of TLR8 p53-SNP rs3761624 (A/G*) relative to the TSS of the gene and to the p53 Response Element (p53RE). Blinded TLR8 gene (B) and protein (C) expression and (D) p53 occupancy profiles in human lymphocytes after 24 hours of treatment with p53 activators nutlin (10 μM), DXR (1 μM), and IR (4 Gy). Each dot represents a different donor. A total of 27 donors were evaluated for gene and protein expression, and 17 for occupancy. Presented in (E) nutlin, (F) DXR, and (G) IR are the decoded 24-hour results for TLR8 mRNA (n = 25) and protein (n = 25) expression profiles and p53 occupancy (n = 16) grouped by rs3761624 A/G genotypes. The horizontal bars represent the mean values. *P < 0.05; **P < 0.01; ***P < 0.0001 (2-tailed unpaired Student’s t test).

Article Snippet: A subset of samples was genotyped for the TLR8 rs rs3761624 SNP using the TaqMan SNP genotyping assay C__27497635_10 by the EPR laboratory.

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: p53-responsive TLR8 SNP enhances human innate immune response to respiratory syncytial virus

doi: 10.1172/JCI128626

Figure Lengend Snippet: (A) IL-6 secretion measured by ELISA in blinded samples from PHA-stimulated lymphocytes pretreated with DMSO, nutlin, DXR, or IR for 24 hours then washed twice with PBS and challenged with water, TLR8 PAMP ssRNA40 (5 μg/mL) or its decoy ssRNA41 (5 μg/mL) for 3 hours. Each dot represents a different donor (n = 27). Presented are IL-6 secretion profiles (n = 25) grouped by A/G rs3761624 genotypes after (B) nutlin, (C) DXR, and (D) IR treatments for 24 hours. #P < 0.001 for drug-treated samples when compared with NT or DMSO treatments after ssRNA40 challenge for each genotype (2-tailed unpaired Student’s t test). *P < 0.01; **P < 0.001; ***P < 0.0001 when compared with drug- and ssRNA40-treated group expressing the A (male) or AA (female) rs3761624 SNP allele (s) (Wilcoxon signed-rank test).

Article Snippet: A subset of samples was genotyped for the TLR8 rs rs3761624 SNP using the TaqMan SNP genotyping assay C__27497635_10 by the EPR laboratory.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: The Journal of Clinical Investigation

Article Title: p53-responsive TLR8 SNP enhances human innate immune response to respiratory syncytial virus

doi: 10.1172/JCI128626

Figure Lengend Snippet: DNA was isolated from whole blood, and infants with mild or severe RSV infection were genotyped for TLR8 rs3761624 SNP. χ2 analysis of RSV disease severity among (A) female and (B) male homozygous WT, heterozygous, and homozygous variants was used to determine association of genotype with increased disease severity. The percentage of patients for each genotype is shown below each bar.

Article Snippet: A subset of samples was genotyped for the TLR8 rs rs3761624 SNP using the TaqMan SNP genotyping assay C__27497635_10 by the EPR laboratory.

Techniques: Isolation, Infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR7/8-mediated activation of human NK cells results in accessory cell-dependent IFN-gamma production.

doi: 10.4049/jimmunol.175.3.1636

Figure Lengend Snippet: FIGURE 1. Expression of TLRs by NK cells. a, RT-PCR was con- ducted on RNA isolated from NKL, NK92, YT, and primary NK cells (n 3) using primers specific for TLR3, TLR7, and TLR8. RNA from PBMCs was used as a positive control. b, Cell surface expression of TLR3 on NKL, NK92, YT, and primary NK cells (n 5) was assessed by flow cytometry using a PE-conjugated anti-TLR3 mAb (filled histograms) and compared with isotype matched controls (empty histograms). c, Western blot analysis of protein extracts from the 293T, 293T-TLR7 (upper panel), 293T-TLR8 (lower panel), NKL, NK92, and YT cell lines. Blots were stained with anti-TLR7 (upper panel) or anti-TLR8 (lower panel) specific Abs.

Article Snippet: Following rehydration, proteins were resuspended in reducing sample buffer (30 mM Tris (pH 6.8), 10% (v/v) glycerol, 1% (w/v) SDS, 0.05% (w/v) bromophenol blue, and 2.5% (v/v) -mercaptoethanol), separated using 7.5% SDS-PAGE, electroblotted, and stained with either anti-TLR7 or anti-TLR8 mAb (both from Imgenex), followed by anti-mouse-HRP (Sigma-Aldrich) using West Pico ECL development.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Positive Control, Cytometry, Western Blot, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR8 Senses Staphylococcus aureus RNA in Human Primary Monocytes and Macrophages and Induces IFN-β Production via a TAK1-IKKβ-IRF5 Signaling Pathway.

doi: 10.4049/jimmunol.1403176

Figure Lengend Snippet: FIGURE 4. S. aureus– and TLR8 ligand–induced IFN-b and IL-12 are antagonized by TLR2 activation. Monocytes were stimulated with HK S. aureus Dlgt (108/ml), ssRNA (pU, 0.75ug/ml), and dsRNA (poly(I:C), 0.75 mg/ml) with or without FSL-1 costimulation (10 ng/ml). The synthetic RNA was delivered as a complex with pL-Arg or cationic lipid (L2K) to examine the stimulatory effect of RNA in the endosomal (pL-Arg) versus the cytosolic (L2K) compartment. After 6 h the supernatants were sampled and assayed for (A) IFN-b and (B) IL-12p70 (mean + SD of triplicates). (C and D) Monocytes were stimulated for with TLR8-specific ligand (CL75) or TLR7-specific ligand (R837) with or without FSL-1 coadministration (100 ng/ml) for 2 h (mean 6 SEM, n = 3). The differences were tested by two-way repeated-measurement ANOVA with a Bonferroni posttest. ***p , 0.001. (E and F) Monocytes were treated for 1 h with Alexa Fluor 488–labeled HK S. aureus Δlgt (1 3 108/ml) and then fixed and stained with (D) anti-TLR8 and (E) control Abs. (F) Quantification of the percentage of TLR8+ S. aureus phagosomes using high-content screening analysis (Scan^R) (mean + SD of triplicates). Nuclei are stained with Hoechst (blue). Scale bar, 10 mm. pI:C, poly(I:C).

Article Snippet: The following Abs were used (typically 2–10 mg/ml or 1:100- to 1:200-fold dilution): anti-human TLR8 (Novus Biologicals, no. NBP177203), rabbit IgG control (Novus Biologicals, no. NB810-56910), antihuman IRF5 mAb (Abcam, no. 10T1), anti-human IRF5 mAb (Abcam, no. ab124792), anti-human IRF5 (Santa Cruz Biotechnology, no. D10), antihuman IRF5 (Sigma-Aldrich, no. HPA046700), anti-human IRF3 XP mAb (Cell Signaling Technology, no. 11904), anti-human IRF3 mAb (D83B9 mAb) (Cell Signaling Technology, no. 4302), anti-human IRF3 (Santa Cruz Biotechnology, FL425, no. sc9082), anti-human p65 XP mAb (Cell Signaling Technology, no. 8242), anti-human p65 A (Santa Cruz Biotechnology, no. sc-109), anti-human IRF1 XP mAb (Cell Signaling Technology, no. 8478), anti-human IRF7 (Cell Signaling Technology, no. 4920; EPR4718, Abcam, no. ab109255; H-246, Santa Cruz Biotechnology, no. sc-9083), anti–phospho-IRF7 (Cell Signaling Technology, no. 5184), anti-human IRF8 (Santa Cruz Biotechnology, C-19, no. sc-6058; SigmaAldrich, no. HPA00253; Cell Signaling Technology, no. 5628), normal goat IgG (Santa Cruz Biotechnology), and rabbit IgG XP control mAb (Cell Signaling Technology).

Techniques: Activation Assay, Labeling, Staining, Control, High Content Screening

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR8 Senses Staphylococcus aureus RNA in Human Primary Monocytes and Macrophages and Induces IFN-β Production via a TAK1-IKKβ-IRF5 Signaling Pathway.

doi: 10.4049/jimmunol.1403176

Figure Lengend Snippet: FIGURE 5. S. aureus induces IFN-b production via TLR8 and IRF5 in MDMs. MDMs were treated with siRNA as indicated. Infection with live S. aureus Δlgt (3 3 107/ml) including bacterial culture medium was done for 3 h and the fold induction of IFN-b and TNF expression by infection was determined with reverse transcription–Q-PCR (mean + SEM, n = 4). The effect of silencing was tested by one-way repeated-measurement ANOVA with a Dunnett multiple comparison test compared with control siRNA. *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: The following Abs were used (typically 2–10 mg/ml or 1:100- to 1:200-fold dilution): anti-human TLR8 (Novus Biologicals, no. NBP177203), rabbit IgG control (Novus Biologicals, no. NB810-56910), antihuman IRF5 mAb (Abcam, no. 10T1), anti-human IRF5 mAb (Abcam, no. ab124792), anti-human IRF5 (Santa Cruz Biotechnology, no. D10), antihuman IRF5 (Sigma-Aldrich, no. HPA046700), anti-human IRF3 XP mAb (Cell Signaling Technology, no. 11904), anti-human IRF3 mAb (D83B9 mAb) (Cell Signaling Technology, no. 4302), anti-human IRF3 (Santa Cruz Biotechnology, FL425, no. sc9082), anti-human p65 XP mAb (Cell Signaling Technology, no. 8242), anti-human p65 A (Santa Cruz Biotechnology, no. sc-109), anti-human IRF1 XP mAb (Cell Signaling Technology, no. 8478), anti-human IRF7 (Cell Signaling Technology, no. 4920; EPR4718, Abcam, no. ab109255; H-246, Santa Cruz Biotechnology, no. sc-9083), anti–phospho-IRF7 (Cell Signaling Technology, no. 5184), anti-human IRF8 (Santa Cruz Biotechnology, C-19, no. sc-6058; SigmaAldrich, no. HPA00253; Cell Signaling Technology, no. 5628), normal goat IgG (Santa Cruz Biotechnology), and rabbit IgG XP control mAb (Cell Signaling Technology).

Techniques: Infection, Expressing, Reverse Transcription, Comparison, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR8 Senses Staphylococcus aureus RNA in Human Primary Monocytes and Macrophages and Induces IFN-β Production via a TAK1-IKKβ-IRF5 Signaling Pathway.

doi: 10.4049/jimmunol.1403176

Figure Lengend Snippet: FIGURE 7. TLR8-induced IRF5 nuclear accumulation is dependent on TAK1 and IKKb, whereas TLR8- and TLR2-induced p65 nuclear accumulation is IKKb-dependent but TAK1-independent. Monocytes were stimulated with CL75 (1 mg/ml) and FSL-1 (10 ng/ml) for 1 h and then fixed and stained for IRF5 and p65 by immunofluorescence. Imaging and quantification of IRF5+ and p65+ nuclei were done with Scan^R. (A) IRF5 and p65 double-stained cells without stimulation. (B) IRF5 and p65 double-stained cells after CL75 stimuli. Scale bar, 30 mm. (C) Analysis of nuclear p65 and IRF5 staining intensity (mean fluorescence intensity [MFI]) after CL75 stimulation. (D–F) Effect of pharmacological inhibitors of TAK1 and IKKb on nuclear accumulation of IRF5 and p65 following CL75 and FSL-1 stimulation. (D) Fraction of IRF5+ nuclei 1 h after CL75 stimulation (mean + SEM). (E) Fraction of p65+ nuclei 1 h after CL75 stimulation. (F) Fraction of p65+ nuclei 1 h after FSL-1 stimulation. The effect of inhibitors was tested by one-way ANOVAwith a Dunnett multiple comparison test against the “no inhibitor” condition. (G and H) Effects of gene silencing of various targets in MDMs on the induction of IRF5 and p65 nuclear translocation by S. aureus. Live S. aureus Δlgt (3 3 107/ml) including bacterial culture medium was added to the macrophages for 3 h. The fraction of positive nuclei from independent experiments is shown (mean + SEM, n = 4). The effect of targeted siRNAs was tested by one-way repeated-measurement ANOVA with a Dunnett multiple comparison test against control siRNA. Nuclei are stained with Hoechst (blue). *p , 0.05, **p , 0.01, ***p , 0.001. SA, S. aureus.

Article Snippet: The following Abs were used (typically 2–10 mg/ml or 1:100- to 1:200-fold dilution): anti-human TLR8 (Novus Biologicals, no. NBP177203), rabbit IgG control (Novus Biologicals, no. NB810-56910), antihuman IRF5 mAb (Abcam, no. 10T1), anti-human IRF5 mAb (Abcam, no. ab124792), anti-human IRF5 (Santa Cruz Biotechnology, no. D10), antihuman IRF5 (Sigma-Aldrich, no. HPA046700), anti-human IRF3 XP mAb (Cell Signaling Technology, no. 11904), anti-human IRF3 mAb (D83B9 mAb) (Cell Signaling Technology, no. 4302), anti-human IRF3 (Santa Cruz Biotechnology, FL425, no. sc9082), anti-human p65 XP mAb (Cell Signaling Technology, no. 8242), anti-human p65 A (Santa Cruz Biotechnology, no. sc-109), anti-human IRF1 XP mAb (Cell Signaling Technology, no. 8478), anti-human IRF7 (Cell Signaling Technology, no. 4920; EPR4718, Abcam, no. ab109255; H-246, Santa Cruz Biotechnology, no. sc-9083), anti–phospho-IRF7 (Cell Signaling Technology, no. 5184), anti-human IRF8 (Santa Cruz Biotechnology, C-19, no. sc-6058; SigmaAldrich, no. HPA00253; Cell Signaling Technology, no. 5628), normal goat IgG (Santa Cruz Biotechnology), and rabbit IgG XP control mAb (Cell Signaling Technology).

Techniques: Staining, Imaging, Comparison, Translocation Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR8 Senses Staphylococcus aureus RNA in Human Primary Monocytes and Macrophages and Induces IFN-β Production via a TAK1-IKKβ-IRF5 Signaling Pathway.

doi: 10.4049/jimmunol.1403176

Figure Lengend Snippet: FIGURE 8. Model of TLR2 and TLR8 activation by S. aureus and cross-regulation of the signaling pathways in human primary monocytes and MDMs. S. aureus lipoproteins activate TLR2, leading to CR3 (CD11b/ CD18) upregulation on the monocyte surface and enhanced phagocytosis. Degradation of S. aureus in the phagolysosome releases bacterial ssRNA, which activates TLR8 signaling. Both TLR2 and TLR8 signal via the adaptor molecule MyD88 in TAK1-dependent and TAK1-independent pathways. The TAK1-independent pathway activates NF-kB p65 nuclear translocation in an IKKb-dependent fashion and is important for proin- flammatory cytokine production. Both TLRs activate MAPKs by the TAK1-dependent pathway, but only TLR8 activates IRF5 nuclear trans- location via a mechanism involving TAK1 and IKKb. IRF5 nuclear ac- cumulation via TLR8 signaling is a specific requirement for IFN-b and IL-12 induction by S. aureus, and it also contributes to TNF production, whereas IL-1 and IL-18 are IRF5-independent. TLR2 activation inhibits TLR8–IRF5 signaling, probably at the level of TAK1/IKKb or upstream.

Article Snippet: The following Abs were used (typically 2–10 mg/ml or 1:100- to 1:200-fold dilution): anti-human TLR8 (Novus Biologicals, no. NBP177203), rabbit IgG control (Novus Biologicals, no. NB810-56910), antihuman IRF5 mAb (Abcam, no. 10T1), anti-human IRF5 mAb (Abcam, no. ab124792), anti-human IRF5 (Santa Cruz Biotechnology, no. D10), antihuman IRF5 (Sigma-Aldrich, no. HPA046700), anti-human IRF3 XP mAb (Cell Signaling Technology, no. 11904), anti-human IRF3 mAb (D83B9 mAb) (Cell Signaling Technology, no. 4302), anti-human IRF3 (Santa Cruz Biotechnology, FL425, no. sc9082), anti-human p65 XP mAb (Cell Signaling Technology, no. 8242), anti-human p65 A (Santa Cruz Biotechnology, no. sc-109), anti-human IRF1 XP mAb (Cell Signaling Technology, no. 8478), anti-human IRF7 (Cell Signaling Technology, no. 4920; EPR4718, Abcam, no. ab109255; H-246, Santa Cruz Biotechnology, no. sc-9083), anti–phospho-IRF7 (Cell Signaling Technology, no. 5184), anti-human IRF8 (Santa Cruz Biotechnology, C-19, no. sc-6058; SigmaAldrich, no. HPA00253; Cell Signaling Technology, no. 5628), normal goat IgG (Santa Cruz Biotechnology), and rabbit IgG XP control mAb (Cell Signaling Technology).

Techniques: Activation Assay, Protein-Protein interactions, Translocation Assay