Journal: PLoS ONE

Article Title: Early decreased TLR2 expression on monocytes is associated with their reduced phagocytic activity and impaired maturation in a porcine polytrauma model

doi: 10.1371/journal.pone.0187404

Figure Lengend Snippet: ( A ). Cells were characterized by CD14 and TLR2 expression by flow cytometry. Blood samples were collected from polytraumatized pigs (n = 12) before trauma as baseline, immediately after surgery/trauma, 3.5 h, 5.5 h, 24 h and 72 h after trauma. The percentage of phagocytizing porcine peripheral monocytes out of total monocytes was determined ( B ). Whole blood samples were incubated with pHrodo® Red S . aureus BioParticles® Conjugate. The measurement was performed by flow cytometry. Monocytes were discriminated by forward and sideward scatter. Data are shown as mean ± sem. *: p <0.05 vs . baseline (before surgery). Data are shown as mean ± sem. *: p <0.05 vs . baseline (before surgery).

Article Snippet: The samples were kept at room temperature and incubated for one hour at room temperature in darkness with the rat IgG polyclonal neutralizing human TLR2 (Invivogen) (20 μg/ml).

Techniques: Expressing, Flow Cytometry, Incubation

Journal: PLoS ONE

Article Title: Early decreased TLR2 expression on monocytes is associated with their reduced phagocytic activity and impaired maturation in a porcine polytrauma model

doi: 10.1371/journal.pone.0187404

Figure Lengend Snippet: Blood samples were collected from healthy pigs (n = 10). Whole blood samples were incubated in the first step with TLR2 neutralizing (nAB TLR2) or control (nAB ctrl) antibody, or without antibody (ctrl). In the next step, the samples were incubated with pHrodo® Red S . aureus BioParticles® Conjugate. The measurement was performed by flow cytometry. Monocytes were discriminated by forward and sideward scatter. Data are shown as mean ± sem. *: p <0.05 vs . ctrl.

Article Snippet: The samples were kept at room temperature and incubated for one hour at room temperature in darkness with the rat IgG polyclonal neutralizing human TLR2 (Invivogen) (20 μg/ml).

Techniques: Incubation, Flow Cytometry

Journal: Immunology

Article Title: Differential regulation of Toll-like receptor signalling in spleen and Peyer's patch dendritic cells

doi: 10.1111/j.1365-2567.2010.03317.x

Figure Lengend Snippet: Gene-specific primers and probe combinations assayed by quantitative real-time polymerase chain reaction

Article Snippet: Flow cytometry Using a FACSCalibur (BD Biosciences, San Jose, CA) flow cytometer, the surface phenotype of cells was determined using four-colour staining with the following antibodies: fluorescein isothiocyanate-conjugated (-FITC) or allophycocyanin-conjugated CD11c (HL3), phycoerythrin-conjugated (-PE) CD80 (16-10A1) (BD-Pharmingen, Oxford, UK); TLR2-PE (6C2), TLR4/MD2-PE-Cy7 (MTS510) (eBioscience, San Diego, CA) and major histocompatibility class II (MHCII)-FITC (M5/114) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Sequencing

Journal: Immunology

Article Title: Differential regulation of Toll-like receptor signalling in spleen and Peyer's patch dendritic cells

doi: 10.1111/j.1365-2567.2010.03317.x

Figure Lengend Snippet: A greater percentage of spleen dendritic cells (DCs) express surface Toll-like receptor 2 (TLR2) and TLR4 than Peyer's patch (PP) DCs. (a) AutoMACS-separated CD11c+ cells from spleen and PP were gated on the expression of CD11c and MHCII as in Fig. 1(a). The expression of surface TLR2 and TLR4-MD2 was then determined on the double-positive population. Values in the upper right of the histograms indicate the mean fluorescence intensity of cells in the indicated gate, n = 3. Bars represent mean ± SEM of percentage of cells falling in the indicated gate of the CD11c+ MHCII+ population, n = 3. (b) autoMACS-separated, flow cytometrically sorted DCs were analysed for gene expression of tlr 1–9. Expression was determined as fold induction compared with β-actin housekeeper. There was no statistical difference between spleen and PP in the messenger RNA levels of any of the genes investigated. Bars represent mean ± SEM, n = 4 or n = 5. Significance determined by unpaired Student's t-test, ***P < 0·001, *P < 0·05.

Article Snippet: Flow cytometry Using a FACSCalibur (BD Biosciences, San Jose, CA) flow cytometer, the surface phenotype of cells was determined using four-colour staining with the following antibodies: fluorescein isothiocyanate-conjugated (-FITC) or allophycocyanin-conjugated CD11c (HL3), phycoerythrin-conjugated (-PE) CD80 (16-10A1) (BD-Pharmingen, Oxford, UK); TLR2-PE (6C2), TLR4/MD2-PE-Cy7 (MTS510) (eBioscience, San Diego, CA) and major histocompatibility class II (MHCII)-FITC (M5/114) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Expressing, Fluorescence, Gene Expression

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: Pre-incubation with directly labeled TLR2 or TLR4 antibodies does not impact on phagocytosis of carboxyfluorescein succinimidyl ester (CSFE)-labeled E. bovis by polymorphonuclear neutrophils (PMN). Isolated PMN (1 × 10 6 per sample; n = 3) were pre-treated with TLR2 and TLR4 antibodies for 30 min and exposed to carboxyfluorescein succinimidyl ester (CFSE)-labeled E. bovis sporozoites (2.5 µm, 30 min) at a 1:1 ratio for two hours for subsequent flow cytometric analysis. Pre-incubation of PMN with antibodies to bovine TLR2 and TLR4 did not seem to impact on the phagocytosis of CFSE-labeled E. bovis sporozoites. Data are represented as the mean of 3 replicates ± SD and were analyzed using an unpaired Student’s t -test using GraphPad Prism V.8.4.3 (GraphPad Software Inc., San Diego, CA, USA).

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Incubation, Labeling, Isolation, Software

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: E. bovis increases TLR2 and TLR4 expression on PMN. PMN (1 × 10 6 per sample; n = 3) were incubated with TLR2 and TLR4 antibodies for 30 min prior to exposure to live E. bovis for two hours for subsequent Flow cytometry analyses (FACS). Incubation of PMN with E. bovis significantly increases TLR2 ( A ) and TLR4 ( B ) expression (**** p < 0.0001). Data are represented as the mean of 3 replicates ± SD and were analyzed using an unpaired Student’s t -test using GraphPad Prism V.8.4.3 (GraphPad Software Inc.). p -value notation; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Expressing, Incubation, Flow Cytometry, Software

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: IL-8 production in PMN upon E. bovis exposure. Supernatants of PMN (1 × 10 6 per sample; n = 3) treated with TLR2 and TLR4 antibodies and exposed to live E. bovis sporozoites (1:1 ratio; 2 h) were assessed for the presence of IL-8 by ELISA analysis. TLR2-treated PMN exposed to sporozoites showed a significant increase in IL-8 production (* p < 0.05) when compared to PMN in media. Likewise, a significant increase in IL-8 production (** p < 0.01) was observed in the same experimental condition when compared to the respective control without exposure to E. bovis. Data are represented as the mean of 3 replicates ± SD and were analyzed using an unpaired Student’s t -test using GraphPad Prism V.8.4.3 (GraphPad Software Inc., San Diego, CA, USA). p -value notation; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Software

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: Induction of Toll-like receptor (TLR)-dependent NF-κB activation by E. bovis sporozoites. In order to investigate the activation of TLRs in bovine PMN, we used HEK cells expressing either bovine TLR2 (A) or a combination of bovine TLR4/MD2 (B). Cells were exposed to different E. bovis sporozoite preparations: live, heat killed (HK) or antigen ( Eb Ag) for 24 h and assessed for activation of transcription factor NF-κB using a luciferase reporter assay. ( A ) HK sporozoites and Eb Ag induced substantial TLR2-dependent NF-κB activation compared to media alone (*** p < 0.0001, ** p < 0.01 and * p < 0.05, respectively). TLR2-induced NF-κB significantly increases when exposed to Eb Ag compared to live E. bovis ( p < 0.05). ( B ) HK sporozoites induced a significant NF-κB response when compared to media ( p < 0.05) and when compared to live parasitic stages ( p < 0.01). A significant increase in NF-κB response was observed in Eb Ag when compared to media ( p < 0.05). In both experiments, Pam 3 CSK 4 and Lipopolysaccharides (LPS) served as ligand controls for TLR2 and TLR4/MD2, respectively, and phorbol 12-myristate 13-acetate (PMA) was used as an NF-κB technical control (data not shown for clarity). Data are represented as the mean of 3 replicates ± SD and were analyzed using an unpaired Student’s t -test using GraphPad Prism V.8.4.3 (GraphPad Software Inc.).

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Activation Assay, Expressing, Luciferase, Reporter Assay, Control, Software

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: Immunofluorescence analysis on bovine PMN activation of TLR2 and TLR 4 by E. bovis and concomitant neutrophil extracellular trap (NET) formation. PMN ( n = 3; 5 × 10 5 ) were exposed to vital E. bovis sporozoites (ratio 1:1) on poly- l -lysine-treated coverslips (120 min, 37 °C) and fixed for further antibody exposure (60 min) with anti-TLR2 ( C ) and anti-TLR4 ( D ) antibodies and anti-histone H1, H2A/H2B, H3, H4 antibody ( E , F ). Coverslips were mounted with ProLong Antifade containing DAPI ( A , B ) which was used for observation of PMN nuclei and NET extracellular DNA by fluorescence microscopy analysis. In both cases, expression of TLR2 and TLR4 was observed on the surface of bovine PMN (red) co-localized with NET-derived histones (green) and extracellular DNA (blue), as indicated by yellow arrows (( G , H )—overlay of images collected for nucleic acid, TLR2/TLR and histone staining). Co-localization of TLR-positive signals with early stages of NETosis are indicated by white arrows ( G , H ). TLR-positive signals without NETosis are indicated by orange arrows ( G , H ). Images were visualized by using an inverted Olympus IX81 ® epifluorescence microscope equipped with a digital camera (XM10 ® , Olympus, Tokyo, Japan). Scale bar magnitude: 20 µm.

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Immunofluorescence, Activation Assay, Fluorescence, Microscopy, Expressing, Derivative Assay, Staining

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: Antibodies.

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Recombinant

Journal: Arthritis Research & Therapy

Article Title: Hyaluronic acid fragments enhance the inflammatory and catabolic response in human intervertebral disc cells through modulation of toll-like receptor 2 signalling pathways

doi: 10.1186/ar4274

Figure Lengend Snippet: The effect of gene silencing on fHA-mediated IL-6 production in IVD cells . A ) siRNA-mediated knockdown of genes Toll like receptor ( TLR ) 2 , TLR4 , CD44 and RHAMM was confirmed after 30 hours in intervertebral disc (IVD) cells by qRT-PCR ( n = 4). In each case, gene expression was calculated as fold change as compared to untreated cells. The use of a non-specific scrambled siRNA (siRNA (S)), confirmed specificity of gene knockdown. B, C ) Interleukin (IL)-6 protein production by Pam3CSK4- (25 ng/ml) ( n = 4) ( B ) and lipopolysaccharide (LPS)- (25 ng/ml) ( n = 3). ( C ) stimulated IVD cells following gene knockdown of TLR2 or TLR4 respectively, as determined by IL-6 ELISA. ( D ) hyaluronic acid fragment (fHA)-treated (20 μg/ml) IVD cells following gene knockdown as determined by IL-6 ELISA ( n = 4). IL-6 protein levels are represented as a percentage of those measured for untreated cells. In all cases, analyses were performed in triplicate and values expressed as mean ± S.D. Statistical analysis was performed using the Student's t -test, * P <0.01.

Article Snippet: Cells were then pre-incubated for one hour with either an affinity purified polyclonal rat anti-human TLR2 neutralizing antibody (final concentration 5 μg/ml) (LabForce, Switzerland) or an isotype matched IgG control (Lucerna-Chem, Luzern, Switzerland).

Techniques: Knockdown, Quantitative RT-PCR, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: Hyaluronic acid fragments enhance the inflammatory and catabolic response in human intervertebral disc cells through modulation of toll-like receptor 2 signalling pathways

doi: 10.1186/ar4274

Figure Lengend Snippet: The effect of TLR2 inhibition on fHA-mediated IL-6 production in IVD cells . Interleukin (IL)-6 protein production by Pam3CSK4- (25 ng/ml) ( A ) and hyaluronic acid fragment (fHA)-treated (20 μg/ml) ( B ) intervertebral disc (IVD) cells following antibody-mediated neutralization of Toll like receptor (TLR)2 activity ( n = 4). IL-6 protein levels are represented as a percentage of those measured for untreated cells. In all cases, analyses were performed in triplicate and values expressed as mean ± S.D. Statistical analysis was performed using the Student's t -test, * P <0.05 as compared to cells treated with non-specific IgG control antibody.

Article Snippet: Cells were then pre-incubated for one hour with either an affinity purified polyclonal rat anti-human TLR2 neutralizing antibody (final concentration 5 μg/ml) (LabForce, Switzerland) or an isotype matched IgG control (Lucerna-Chem, Luzern, Switzerland).

Techniques: Inhibition, Neutralization, Activity Assay, Control

Journal: Gut Microbes

Article Title: ADP-heptose attenuates Helicobacter pylori -induced dendritic cell activation

doi: 10.1080/19490976.2024.2402543

Figure Lengend Snippet: TLR2 mediated Helicobacter pylori uptake is essential for potent dendritic cell activation. (a) TLR2 mRNA expression upon stimulation with H. pylori wt or mutant (Δ) (MOI 5) at indicated time points. Mean±SD of three individual donors is shown. (b) TLR2 surface expression was monitored by flow cytometry 16 h post-infection (MOI 5) ( n = 4). (c,d) H. pylori strains were stained with eFluor670 proliferation dye prior to infection. One hour post infection with H. pylori wt or the ADP-heptose-deficient mutant (Δ) (MOI 5), DCs were subjected to immunofluorescence and stained for CD45, DAPI and TLR2. Internalization of bacteria as well as TLR2 localization was analyzed by confocal fluorescence microscopy. Orthogonal views of confocal z-stacks of one out of three representative donors are shown. Scale bar: 5 µm. (e) DCs were treated with a TLR2 neutralizing antibody 20 min prior to infection. After 1 h of infection with eFluor670 stained H. pylori (MOI 5), DCs were subjected to immunofluorescence and stained for CD45 and DAPI. Maximum intensity projections of confocal z-stacks of one representative out of three donors are shown. Scale bar: 5 µm. (f,g) DCs were treated with a TLR2 neutralizing antibody 20 min prior to infection with H. pylori (wt) or the ADP-heptose deficient mutant (Δ) at an MOI of 5. After 16 h CD40 expression (f) and IL-12p70 secretion (g) was monitored by flow cytometry and multiplex assay ( n = 6). For statistical analysis one-way ANOVA with a šídák’s post-hoc test was performed.

Article Snippet: Following primary antibodies were used: mouse anti-human CD45 (ab30470 Abcam, 1:200), rabbit anti-human TLR2 (NB100–56720 Novus Biologicals, 1:400).

Techniques: Activation Assay, Expressing, Mutagenesis, Flow Cytometry, Infection, Staining, Immunofluorescence, Bacteria, Fluorescence, Microscopy, Multiplex Assay

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

Article Title: SHP-1 As a Critical Regulator of Mycoplasma pneumoniae -Induced Inflammation in Human Asthmatic Airway Epithelial Cells

doi: 10.4049/jimmunol.1100573

Figure Lengend Snippet: M. pneumoniae infection induced TLR2–SHP-1 dynamic association. (A) Immunoblot analysis of baseline TLR2 expression in airway epithelial cells from nonasthmatic and asthmatic subjects. The data representing the percentage of densitometry values of nonasthmatic cells are expressed as mean ± SEM (right panel). (B) TLR2-associated SHP-1 levels in airway epithelial cells was measured by coimmunoprecipitation. TLR2 was immunoprecipitated from airway epithelial cell lysates of nonasthmatic and asthmatic subjects without mycoplasma infection (0 h), infected with mycoplasma for 1 and 2 h. TLR2 immunoprecipitates were blotted with anti-TLR2 (top left panel) and anti–SHP-1 (bottom left panel) Ab. TLR2-associated SHP-1 at 2 h after M. pneumoniae infection is expressed as fold change of densitometry values from nonasthmatic cells without mycoplasma treatment (0 h) and was increased in nonasthmatic cells compared with asthmatic cells. The data are presented as mean ± SEM (n = 5). *p < 0.01 compared with asthmatic group.

Article Snippet: The membranes were probed with rabbit anti-human TLR2 Ab (IMGENEX, San Diego, CA), mouse anti-human SHP-1 (Santa Cruz Biotechnology), or a Ser 473 phosphospecific Ab to Akt (Cell Signaling, Danvers, MA) overnight at 4°C.

Techniques: Infection, Western Blot, Expressing, Immunoprecipitation

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

Article Title: SHP-1 As a Critical Regulator of Mycoplasma pneumoniae -Induced Inflammation in Human Asthmatic Airway Epithelial Cells

doi: 10.4049/jimmunol.1100573

Figure Lengend Snippet: Proposed model for the mechanism of SHP-1–mediated inhibition of M. pneumoniae-activated TLR2 signaling in normal and asthmatic airway epithelial cells. The ligation of TLR2 by M. pneumoniae initiates TLR2-mediated proinflammatory signaling pathway, resulting in the production of IL-8. M. pneumoniae binding to TLR2 activates and recruits SHP-1, which inhibits the nuclear translocation of NF-κB directly or through inhibition of PI3K/Akt abrogating NF-κB activation and subsequently prevents IL-8 production. In addition, the nuclear SHP-1 may also inhibit NF-κB function by certain nuclear mediators (left panel). In asthmatic airway epithelial cells, M. pneumoniae-induced SHP-1 activation is defective, which contributes to the increased activation of PI3K/Akt and NF-κB, as well as abundant IL-8 production (right panel).

Article Snippet: The membranes were probed with rabbit anti-human TLR2 Ab (IMGENEX, San Diego, CA), mouse anti-human SHP-1 (Santa Cruz Biotechnology), or a Ser 473 phosphospecific Ab to Akt (Cell Signaling, Danvers, MA) overnight at 4°C.

Techniques: Inhibition, Ligation, Binding Assay, Translocation Assay, Activation Assay