Journal: Applied and Environmental Microbiology

Article Title: Piliation of Lactobacillus rhamnosus GG Promotes Adhesion, Phagocytosis, and Cytokine Modulation in Macrophages

doi: 10.1128/AEM.03949-14

Figure Lengend Snippet: Role of CD11b in adhesion and uptake of L. rhamnosus GG by RAW 264.7 cells and effect of TLR2 in the subsequent immunomodulatory response. (A) Role of CD11b in adhesion of the mutants to macrophages (left) and phagocytosis of the mutants by macrophages (right) relative to the results for the wild-type strain, which were set equal to 1. Bacterial strains (5 × 107 CFU/ml) were coincubated for 1 h with 1 × 106 RAW 264.7 cells that had previously been treated with anti-mouse CD11b monoclonal antibody (5 μg/ml) for 30 min. The data are presented as the mean relative adhesion or phagocytosis ± standard deviation. These experiments were done in triplicate. Asterisks represent statistically significant differences compared to the results for the controls (no antibody treatment). (B) The effect of TLR2 on the relative levels of IL-10 and IL-6 mRNA expression by RAW 264.7 macrophages was analyzed by pretreatment of macrophages with antibodies against TLR2 (5 to 10 μg/ml) for 30 min and then coincubation with L. rhamnosus GG strains for 3 h. Bacteria were administered at a ratio of 1:50 of cells to bacteria. The results are mean values ± standard deviations from three separate experiments. The values are normalized against those for GAPDH. Asterisks denote statistically significant differences from the results for the control (no antibody treatment).

Article Snippet: The blocking monoclonal anti-human/mouse CD282 TLR2 purified antibody (clones T2.5 and 6C2) and monoclonal anti-mouse CD11b purified antibody (clone M1/70) were obtained from eBioscience (San Diego, CA, USA).

Techniques: Standard Deviation, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex *

doi: 10.1074/jbc.M113.453266

Figure Lengend Snippet: Recombinant sTLR proteins are monomeric, properly folded, and biologically functional. A, 100 μg of each purified sTLR protein as indicated was loaded for 7.5% SDS-PAGE and stained with Coomassie blue dye. B, each purified sTLR protein as indicated was analyzed by size exclusion chromatography using a Superdex 200 column. C, soluble TLRs were incubated in microtiter plate wells coated with either the anti-TLR1 mAb (clone GD2F4) or the anti-TLR2 mAb (T2.5) as indicated. Binding of soluble TLR1, TLR1P315L, or TLR2 was detected using HRP-conjugated anti-HA and anti-FLAG mAbs. D, SW620 cells were co-transfected with full-length TLR1, TLR2, an IL-8 promoter-driven luciferase reporter gene, and a Renilla luciferase transfection control. 48 h post-transfection, the cells were stimulated with 10 ng/ml Pam3CSK4 with or without 1 μg/ml soluble TLR1 or TLR2 as indicated. Firefly luciferase activities were normalized to that of the Renilla luciferase control. These values were normalized to that of empty CMV vector whose value was taken as 1. Error bars represent the S.D. of three independent events. mAu, milliabsorbance units.

Article Snippet: The unconjugated anti-human TLR1 mAb (clone GD2.F4, CD281) and anti-human TLR2 mAb (clone T2.5, CD282) were obtained from eBioscience (San Diego, CA).

Techniques: Recombinant, Functional Assay, Purification, SDS Page, Staining, Size-exclusion Chromatography, Incubation, Binding Assay, Transfection, Luciferase, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex *

doi: 10.1074/jbc.M113.453266

Figure Lengend Snippet: LBP and soluble CD14 enhance soluble TLR1·TLR2·Pam3CSK4 ternary complex formation but are not part of the final ternary complex. In A–D, various combinations of 0.25 μm TLR1, 0.05 μm TLR2, 2.5 μm Pam3CSK4, 0.05 μm LBP, and/or 0.25 μm sCD14 were incubated for 2 h at 37 °C in a 500-μl volume of PBS buffer, pH 7.4 as indicated. Protein complexes were separated by size exclusion chromatography. The expected molecular weight of the TLR monomers and dimers was estimated by column calibration using known molecular weight standards. Proteins in eluted fractions were separated by 7.5% SDS-PAGE and transferred by Western blotting, and TLR1, TLR2, LBP, and sCD14 were detected using suitable antibodies and HRP conjugates (see “Experimental Procedures”). The results shown are representative of at least three independent experiments. mAu, milliabsorbance units.

Article Snippet: The unconjugated anti-human TLR1 mAb (clone GD2.F4, CD281) and anti-human TLR2 mAb (clone T2.5, CD282) were obtained from eBioscience (San Diego, CA).

Techniques: Incubation, Size-exclusion Chromatography, Molecular Weight, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex *

doi: 10.1074/jbc.M113.453266

Figure Lengend Snippet: Soluble TLRs do not form homodimers when incubated with Pam3CSK4. 0.5 μm TLR1 (A), 0.5 μm TLR2 (B), a 0.25 μm concentration each of TLR1 and TLR2 (C), or a 0.25 μm concentration each of TLR1P315L and TLR2 (D) were preincubated with a 5-fold molar excess of Pam3CSK4 (2.5 μm) together with 0.05 μm LBP and 0.25 μm sCD14 in PBS, pH 7.4 buffer for 2 h at 37 °C in a 500-μl reaction volume. Proteins and protein complexes were separated and analyzed as described in the legend of Fig. 2. The results shown are representative of at least three independent experiments. mAu, milliabsorbance units.

Article Snippet: The unconjugated anti-human TLR1 mAb (clone GD2.F4, CD281) and anti-human TLR2 mAb (clone T2.5, CD282) were obtained from eBioscience (San Diego, CA).

Techniques: Incubation, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex *

doi: 10.1074/jbc.M113.453266

Figure Lengend Snippet: LBP and soluble CD14 enhance complex formation between TLR1, TLR2, and the OspA lipoprotein of B. burgdorferi. In A–E, various combinations of 0.25 μm TLR1, 0.25 μm TLR2, 1.25 μm OspA, 0.05 μm LBP, and/or 0.25 μm sCD14 were incubated for 2 h at 37 °C in a 500-μl volume of PBS buffer, pH 7.4 as indicated. Proteins and protein complexes were separated and analyzed as described in the legend of Fig. 2. The results shown are representative of at least three independent experiments. mAu, milliabsorbance units.

Article Snippet: The unconjugated anti-human TLR1 mAb (clone GD2.F4, CD281) and anti-human TLR2 mAb (clone T2.5, CD282) were obtained from eBioscience (San Diego, CA).

Techniques: Incubation

Journal: The Journal of Biological Chemistry

Article Title: Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex *

doi: 10.1074/jbc.M113.453266

Figure Lengend Snippet: Either LBP or soluble CD14 can independently enhance TLR1·TLR2·Pam3CSK4 ternary complex formation. 0.25 μm TLR1, 0.25 μm TLR2, and 2.5 μm Pam3CSK4 were incubated with 0.25 μm BSA (A), 0.05 μm LBP (B), and 0.25 μm sCD14 (C) for 2 h at 37 °C in a 500-μl volume of PBS, pH 7.4 buffer. Proteins and protein complexes were separated and analyzed as described in the legend of Fig. 2. The results shown are representative of at least three independent experiments. mAu, milliabsorbance units.

Article Snippet: The unconjugated anti-human TLR1 mAb (clone GD2.F4, CD281) and anti-human TLR2 mAb (clone T2.5, CD282) were obtained from eBioscience (San Diego, CA).

Techniques: Incubation

Journal: The Journal of Biological Chemistry

Article Title: Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex *

doi: 10.1074/jbc.M113.453266

Figure Lengend Snippet: Substoichiometric concentrations of either LBP or soluble CD14 are sufficient to enhance TLR1·TLR2·Pam3CSK4 ternary complex formation. 0.25 μm TLR1, 0.25 μm TLR2, and 2.5 μm Pam3CSK4 were incubated with various concentrations (250, 50, 10, 5, and 0 nm) of either LBP (left panel) or sCD14 (right panel) for 2 h in a 500-μl volume at 37 °C in PBS buffer, pH 7.4. Proteins and protein complexes were separated and analyzed as described in the legend of Fig. 2. The results shown are representative of at least three independent experiments. mAu, milliabsorbance units.

Article Snippet: The unconjugated anti-human TLR1 mAb (clone GD2.F4, CD281) and anti-human TLR2 mAb (clone T2.5, CD282) were obtained from eBioscience (San Diego, CA).

Techniques: Incubation

Journal: The Journal of Biological Chemistry

Article Title: Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex *

doi: 10.1074/jbc.M113.453266

Figure Lengend Snippet: Either LBP or soluble CD14 enhances cellular responses to Pam3CSK4 and OspA. HEK 293F cells were co-transfected with vectors expressing full-length TLR1 and TLR2 or empty CMV control vector as indicated together with an NF-κB-promoter driven luciferase reporter gene and a Renilla luciferase reporter gene. About 48 h post-transfection, cells were stimulated with 1 ng/ml Pam3CSK4, OspA, or the non-acylated Ac2CSK4 control in the presence of 0.1 μg/ml LBP, sCD14, or human serum albumin (HSA) as indicated (left side). In one set of experiments, agonists were preincubated with proteins for 1 h at 37 °C prior to addition to transfected cells (right side). Cell values on the y axis represent the level of constitutive reporter activation normalized to the empty CMV vector control (value of 1). Error bars represent the S.D. of three independent values.

Article Snippet: The unconjugated anti-human TLR1 mAb (clone GD2.F4, CD281) and anti-human TLR2 mAb (clone T2.5, CD282) were obtained from eBioscience (San Diego, CA).

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Activation Assay

Journal: Cellular microbiology

Article Title: Toll-like receptors 1 and 2 cooperatively mediate immune responses to curli, a common amyloid from enterobacterial biofilms

doi: 10.1111/j.1462-5822.2010.01485.x

Figure Lengend Snippet: (A) THP-1 cells were stimulated with purified curli fibrils (black bars), synthetic tri-acylated lipopeptide (Pam3CSK4) (gray bars), or synthetic CsgA111-151 peptide (open bars) in the presence or absence (No Ab) of blocking antibodies against TLR2 (anti-TLR2), TLR1 (anti-TLR1) or a combination of anti-TLR1 and anti-TLR2 antibodies (anti-TLR1/TLR2). Mock-treated THP-1 cells served as a negative control. (B) THP-1 cells were stimulated with LPS (gray bars) in the presence or absence of blocking antibodies against TLR4 (anti-TLR4), TLR2 or TLR1. Expression of IL8 in (A) and (B) was determined by quantitative real-time PCR. Data are expressed as fold increases over mRNA levels detected in mock-treated cells. Bars represent averages from at least three independent experiments ± standard error.

Article Snippet: Blocking anti-human TLR2 (T2.5) and TLR1 (GD2.F4), antibodies were purchased from eBioscience.

Techniques: Purification, Blocking Assay, Negative Control, Expressing, Real-time Polymerase Chain Reaction

Journal: Cellular microbiology

Article Title: Toll-like receptors 1 and 2 cooperatively mediate immune responses to curli, a common amyloid from enterobacterial biofilms

doi: 10.1111/j.1462-5822.2010.01485.x

Figure Lengend Snippet: (A) Nos2 mRNA levels observed in the liver 8 hours after intraperitoneal infection of wild type mice (black bars) or TLR2-deficient mice (open bars) with 108 colony forming units (CFU) of the indicated E. coli strains. Each bar represents the average fold increases in Nos2 expression of E. coli-infected mice (n=4) compared to mock-infected mice (n=4) ± standard error. Statistical significance of differences is indicated by a bracket. (B) Bacterial numbers recovered from the liver 8 hours after intraperitoneal infection of wild type mice (black bars) or TLR2 deficient mice (open bars) with 108 colony forming units (CFU) of the indicated E. coli strains.

Article Snippet: Blocking anti-human TLR2 (T2.5) and TLR1 (GD2.F4), antibodies were purchased from eBioscience.

Techniques: Infection, Expressing