Journal: PLoS ONE

Article Title: Functional Effects of Toll-Like Receptor (TLR)3, 7, 9, RIG-I and MDA-5 Stimulation in Nasal Epithelial Cells

doi: 10.1371/journal.pone.0098239

Figure Lengend Snippet: Sections of nasal biopsies were incubated with antibodies against TLR3 ( A ), TLR7 ( B ), TLR9 ( C ), RIG-I ( D ), and MDA-5 ( E ) and visualized by 3, 3′-diaminobenzidine (brown). In control slides ( F ), N-series universal negative control reagent was used. All sections were accompanied with a square magnification. All slides were counterstained with haematoxylin (blue). The figure shows one representative biopsy out of four (3 male, 1 female). The arrows indicate positive stained cells.

Article Snippet: The nasal biopsy sections were incubated at 4°C overnight and the epithelial cells at room temperature (RT) for 1 hour with monoclonal mouse anti-human Abs against TLR3 (Cat. no. 40C1285.6; AMS Biotechnology, Abingdon, UK), diluted 1∶20, TLR9 (211-MG-1TLR9; Acris antibodies, Hiddenhausen, Germany), diluted 1∶40 and RIG-I (ab77010; Abcam, Cambridge, UK), diluted 1∶20, or polyclonal rabbit anti-human Abs against TLR7 (ab45371), diluted 1∶20, and MDA-5 (ab69983; Abcam), diluted 1∶40.

Techniques: Incubation, Negative Control, Staining

Journal: PLoS ONE

Article Title: Functional Effects of Toll-Like Receptor (TLR)3, 7, 9, RIG-I and MDA-5 Stimulation in Nasal Epithelial Cells

doi: 10.1371/journal.pone.0098239

Figure Lengend Snippet: Epithelial cells from primary HNEC ( A–E ), Detroit-562 ( G–K ) and FaDu ( M–Q ) were incubated with antibodies against TLR3, TLR7, TLR9, RIG-I, and MDA-5 and visualized by 3, 3′-diaminobenzidine (brown). In controls, N-series universal negative control reagent was used ( F, L, R ). All cells were counterstained with haematoxylin (blue). The figure shows one representative staining out of three independent experiments. The markers in the figure are 50 µm.

Article Snippet: The nasal biopsy sections were incubated at 4°C overnight and the epithelial cells at room temperature (RT) for 1 hour with monoclonal mouse anti-human Abs against TLR3 (Cat. no. 40C1285.6; AMS Biotechnology, Abingdon, UK), diluted 1∶20, TLR9 (211-MG-1TLR9; Acris antibodies, Hiddenhausen, Germany), diluted 1∶40 and RIG-I (ab77010; Abcam, Cambridge, UK), diluted 1∶20, or polyclonal rabbit anti-human Abs against TLR7 (ab45371), diluted 1∶20, and MDA-5 (ab69983; Abcam), diluted 1∶40.

Techniques: Incubation, Negative Control, Staining

Journal: PLoS ONE

Article Title: Functional Effects of Toll-Like Receptor (TLR)3, 7, 9, RIG-I and MDA-5 Stimulation in Nasal Epithelial Cells

doi: 10.1371/journal.pone.0098239

Figure Lengend Snippet: HNEC ( A ), Detroit-562 ( B ) and FaDu ( C ) were stained intracellularly with Abs against TLR3, TLR7, TLR9, RIG-I and MDA-5 (open histograms) or appropriate isotype control (shaded histograms) and analyzed by flow cytometry. Representative pictures from one out of three independent experiments are shown.

Article Snippet: The nasal biopsy sections were incubated at 4°C overnight and the epithelial cells at room temperature (RT) for 1 hour with monoclonal mouse anti-human Abs against TLR3 (Cat. no. 40C1285.6; AMS Biotechnology, Abingdon, UK), diluted 1∶20, TLR9 (211-MG-1TLR9; Acris antibodies, Hiddenhausen, Germany), diluted 1∶40 and RIG-I (ab77010; Abcam, Cambridge, UK), diluted 1∶20, or polyclonal rabbit anti-human Abs against TLR7 (ab45371), diluted 1∶20, and MDA-5 (ab69983; Abcam), diluted 1∶40.

Techniques: Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Functional Effects of Toll-Like Receptor (TLR)3, 7, 9, RIG-I and MDA-5 Stimulation in Nasal Epithelial Cells

doi: 10.1371/journal.pone.0098239

Figure Lengend Snippet: Nasal biopsies and epithelial cells were cultured in the absence (Untreated) or presence of 10 µg/ml poly(I:C) (TLR3), 10 µg/ml R-837 (TLR7), 1 µM CpG (TLR9) and 1 µg/ml poly(I:C)/LyoVec (RIG-I/MDA-5). TNF-α (10 ng/ml) was used as a positive control (data not shown). After 24 h, supernatants from nasal biopsies (n = 5) ( A–D ), HNEC (n = 6–9) ( E–H ), Detroit-562 (n = 5) ( I–L ) and FaDu (n = 5–9) ( M–P ) were collected and analyzed for levels of IL-6, IL-8, GM-CSF and IFN-β using ELISA. Data is presented as mean ± SEM of 5 to 9 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: The nasal biopsy sections were incubated at 4°C overnight and the epithelial cells at room temperature (RT) for 1 hour with monoclonal mouse anti-human Abs against TLR3 (Cat. no. 40C1285.6; AMS Biotechnology, Abingdon, UK), diluted 1∶20, TLR9 (211-MG-1TLR9; Acris antibodies, Hiddenhausen, Germany), diluted 1∶40 and RIG-I (ab77010; Abcam, Cambridge, UK), diluted 1∶20, or polyclonal rabbit anti-human Abs against TLR7 (ab45371), diluted 1∶20, and MDA-5 (ab69983; Abcam), diluted 1∶40.

Techniques: Cell Culture, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of Innate Immunity

Article Title: Attenuated Activation of Macrophage TLR9 by DNA from Virulent Mycobacteria

doi: 10.1159/000142731

Figure Lengend Snippet: Human monocyte-derived macrophages express TLR9. THP-1 cells were differentiated by PMA (a) and primary monocytes were differentiated into macrophages by M-CSF treatment (b). RT-PCR and real-time PCR: RNA isolation, reverse transcription and qualitative RT-PCR for TLRs1–10 as well as quantitative real-time PCR for TLR2, TLR4 and TLR9 were performed as described in Materials and Methods. Qualitative RT-PCR was performed in the presence (+) or absence (–) of cDNA. Reaction products of qualitative RT-PCR reactions and a size marker (M) were electrophoresed in agarose and stained with ethidium bromide. Expression levels of TLR2, TLR4 and TLR9 are expressed as mRNA copies of the respective gene per 1,000 copies of GAPDH. Quantification was performed by comparing starting amounts of cDNAs with a standard curve of amplicon cloned into pGEM-T Easy. Data represent means ± SEM of cells from 5–7 donors. Western blot: COS-1 cells were transfected with a pcDNA3.1 vector encoding TLR9 or a vector control. Transfected cells as well as human macrophages were lysed. TLR9 was detected by Western blot by a custom antibody and visualized by an IRDye 800 conjugated anti-mouse IgG. Actin served as a loading control. FACS: Primary macrophages and differentiated THP-1 cells were stained with a custom antibody against TLR9 followed by detection with a secondary antibody via biotin/streptavidin/Cy5 and an FITC-labelled commercial antibody against CD14 as described in Materials and Methods. Cells were analysed by flow cytometry. Data show 1 representative of 3 independent experiments with cells from different donors.

Article Snippet: The membranes were blocked in blocking buffer for near-infraded Western blotting (Rockland) overnight, incubated for 3 h with mouse anti-human TLR9 (clone 2-1E2), washed 3 times, and incubated with IRDye 800 conjugated anti-mouse IgG (Rockland) for 1.5 h. After washing, blots were scanned with an Odyssey Infrared Imaging System (LI-COR Biosciences).

Techniques: Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Isolation, Marker, Staining, Expressing, Amplification, Clone Assay, Western Blot, Transfection, Plasmid Preparation, Flow Cytometry

Journal: Journal of Innate Immunity

Article Title: Attenuated Activation of Macrophage TLR9 by DNA from Virulent Mycobacteria

doi: 10.1159/000142731

Figure Lengend Snippet: Human alveolar macrophages express TLR9 mRNA and protein. TLR mRNA expression was analysed in human alveolar macrophages by qualitative (a) and quantitative RT-PCR (b) and TLR9 protein was detected by immunocytochemistry and Western blot (c). a, b RNA isolation, reverse transcription and qualitative RT-PCR for TLR2 and TLR9 as well as quantitative PCR for TLR2, TLR4 and TLR9 were performed as described in Materials and Methods. Qualitative RT-PCR was performed in the presence (+) or absence (–) of cDNA. Reaction products of qualitative RT-PCR reactions and a size marker (M) were electrophoresed and stained with ethidium bromide. Expression levels of TLR2, TLR4 and TLR9 are expressed as mRNA copies of the respective gene per 1,000 copies of GAPDH. Quantification was performed by comparing starting amounts of cDNAs with a standard curve of amplicon cloned into pGEM-T Easy. c Alveolar macrophages were stained for TLR9 (blue) and with propidium iodide (red) as described in Materials and Methods employing a custom-made antibody and a secondary antibody detected via biotin/streptavidin-Cy5. Stained cells were analysed by confocal microscopy and laser scanning cytometry. A laser scanning cytometer equipped with an argon-ion air-cooled laser and an HeNe laser equipped with a digital camera were used to measure Cy5 fluorescence. Nuclear propidium iodide fluorescence was used as the contouring parameter. Alveolar macrophages for the different analyses were each obtained from different donors. For Western blots, PMA-differentiated THP-1 macrophages or alveolar macrophages were lysed. TLR9 was detected by Western blot by a custom antibody and visualized by an IRDye 800 conjugated anti-mouse IgG. Actin served as a loading control.

Article Snippet: The membranes were blocked in blocking buffer for near-infraded Western blotting (Rockland) overnight, incubated for 3 h with mouse anti-human TLR9 (clone 2-1E2), washed 3 times, and incubated with IRDye 800 conjugated anti-mouse IgG (Rockland) for 1.5 h. After washing, blots were scanned with an Odyssey Infrared Imaging System (LI-COR Biosciences).

Techniques: Expressing, Quantitative RT-PCR, Immunocytochemistry, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Marker, Staining, Amplification, Clone Assay, Confocal Microscopy, Cytometry, Fluorescence

Journal: Journal of Innate Immunity

Article Title: Attenuated Activation of Macrophage TLR9 by DNA from Virulent Mycobacteria

doi: 10.1159/000142731

Figure Lengend Snippet: Altered cytokine production in TLR9-deficient animals infected with virulent or attenuated mycobacteria. Wild-type (BL/6) or TLR9CpG1/CpG1 mice (CpG1) were infected with M. tuberculosis H37Rv or H37Ra. Lungs were homogenized on day 0 or day 18 and TNF-α, IL-1β, IFN-γ and IL-6 levels were measured by a Luminex bead assay (Upstate Beadlyte). Data show means ± SEM of lungs from 4 animals. * p < 0.05 statistically different from values in wild-type animals.

Article Snippet: The membranes were blocked in blocking buffer for near-infraded Western blotting (Rockland) overnight, incubated for 3 h with mouse anti-human TLR9 (clone 2-1E2), washed 3 times, and incubated with IRDye 800 conjugated anti-mouse IgG (Rockland) for 1.5 h. After washing, blots were scanned with an Odyssey Infrared Imaging System (LI-COR Biosciences).

Techniques: Infection, Luminex

Journal: Clinical and Experimental Immunology

Article Title: Patients with tumour necrosis factor (TNF) receptor‐associated periodic syndrome (TRAPS) are hypersensitive to Toll‐like receptor 9 stimulation

doi: 10.1111/cei.13306

Figure Lengend Snippet: Visual depiction of findings: the graphic represents elements of the common pathways active by both tumour necrosis factor receptor 1 (TNF‐R1) signalling and signalling through Toll‐like receptor (TLR)‐9. Arrows indicate activation route of molecules in the pathways. Red arrows indicate those pathways which are found to be both more abundant and more activated as a result of the C33Y tumour necrosis factor receptor‐associated periodic syndrome (TRAPS) mutation and also further stimulated by TLR‐9 ligation as ODN2006 as an artificial activator. Molecules depicted in grey were not studied.

Article Snippet: Following washing with PBA (PBS/1% BSA/0·05% NaN 3 ), the PBMCs were permeabilized using saponin buffer and stained for 2 h in the dark with anti‐human CD289 (TLR‐9) phycoerythrin (PE) or appropriate isotype controls (rat IgG2a PE) (e‐Bioscience/Thermo‐Fisher Scientific).

Techniques: Activation Assay, Mutagenesis, Ligation