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:

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

doi: 10.1172/JCI200420773

Figure Lengend Snippet: Distribution of asialoGM1, TLR2, and TLR4 in infected airway cells. (A) Confocal microscopy was used to image monolayers of polarized 16HBE cells transfected with Flag epitope–tagged TLR2 or TLR4 and stimulated with P. aeruginosa PAO1. z-sections treated with anti-Flag labeled with TRITC (red) and with anti-asialoGM1 labeled with FITC (green) are shown. AsialoGM1 (aGM1) is apical and colocalizes with TLR2 (yellow) in discrete clusters along the apical surface of the monolayers. TLR4 is more diffuse, and colocalization with asialoGM1 is not appreciable. (B) Human airway cells in primary culture isolated from nasal polyps from a CF patient and stimulated with P. aeruginosa PAO1 were stained for asialoGM1 labeled with TRITC and TLR2 labeled with FITC and show abundant colocalization of these receptors.

Article Snippet: Rabbit polyclonal antibodies used were anti–caveolin-1, anti-TLR2, anti–IRAK-1, anti-MYD88, anti-TRAF6, and anti-TLR4 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA); anti-TLR2 antisera from Tularik (South San Francisco, California, USA); and anti-asialoGM1 (Wako, Richmond, Virginia, USA).

Techniques: Infection, Confocal Microscopy, Transfection, FLAG-tag, Labeling, Isolation, Staining

Journal:

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

doi: 10.1172/JCI200420773

Figure Lengend Snippet: Identification of apically exposed components on polarized 16HBE cells exposed to bacteria. (A) Surface-exposed components of the airway cells were biotinylated under control conditions (–) and after a 1-hour exposure to S. aureus (+). After immunoprecipitation with streptavidin, Western hybridizations were done and asialoGM1, TLR2, and caveolin-1 (Cav-1), as well as the kinases IRAK-1 and TRAF6, were detected. (B) Coimmunoprecipitation studies demonstrate TLR2 but not TLR4 in a receptor complex along with asialoGM1. Coimmunoprecipitations of whole-cell lysates from control and S. aureus–stimulated cells were done using anti–caveolin-1, anti-TLR2, and anti-asialoGM1 as capture antibodies with screening for expected components of the TLR pathway, MyD88 and IRAK-1, as well as c-Src and TLR4.

Article Snippet: Rabbit polyclonal antibodies used were anti–caveolin-1, anti-TLR2, anti–IRAK-1, anti-MYD88, anti-TRAF6, and anti-TLR4 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA); anti-TLR2 antisera from Tularik (South San Francisco, California, USA); and anti-asialoGM1 (Wako, Richmond, Virginia, USA).

Techniques: Immunoprecipitation, Western Blot

Journal:

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

doi: 10.1172/JCI200420773

Figure Lengend Snippet: Surface colocalization of MyD88, IRAK-1, and TRAF6 with TLR2 confirmed by confocal microscopy. After stimulation with S. aureus, permeabilized cells were stained with the kinases, each labeled with an Alexa Fluor 488–tagged secondary antibody (green). All were found at the cell surface, colocalized (yellow) with TLR2, labeled with an Alexa Fluor 594–tagged secondary antibody (red).

Article Snippet: Rabbit polyclonal antibodies used were anti–caveolin-1, anti-TLR2, anti–IRAK-1, anti-MYD88, anti-TRAF6, and anti-TLR4 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA); anti-TLR2 antisera from Tularik (South San Francisco, California, USA); and anti-asialoGM1 (Wako, Richmond, Virginia, USA).

Techniques: Confocal Microscopy, Staining, Labeling

Journal:

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

doi: 10.1172/JCI200420773

Figure Lengend Snippet: Lipid rafts are involved in clustering of receptors and signaling. (A) Confocal z-section images demonstrate caveolin-1 labeled with Alexa Fluor 594 (red) and GM1, identified with cholera toxin β-subunit (CTB) conjugated to Alexa Fluor 488 (green), on the apical surfaces of polarized 16HBE cells permeabilized after stimulation with Pam3Cys-Ser-Lys4. (B) CF nasal polyp cells were infected with P. aeruginosa PAO1 (PA) and grown on semipermeable supports, and transmission electron micrograph were obtained after 3 hours of bacterial exposure. Arrow (Cav) indicates the flask-shaped electron-dense structures typical of caveolae (magnification, ∞30,000). (C) Flow cytometry was used to detect superficial caveolin-1 on polarized 16HBE cells after exposure to S. aureus. Unstim, unstimulated. (D) Aliquots of Triton-insoluble lysates of 16HBE cells obtained before (–) and after (+) stimulation with P. aeruginosa PAO1 were fractionated on discontinuous sucrose gradients (4–40%) and were immunoblotted with anti-flotillin, anti-caveolin, anti-TLR-2, anti-IRAK-1, or anti-asialoGM1. Downward arrow indicates raft fraction containing all the components after stimulation. (E) Aliquots of sucrose gradient fractions from cells treated with filipin prior to stimulation with P. aeruginosa PAO1 were immunoblotted with anti-flotillin, anti-TLR2, and anti-asialoGM1.

Article Snippet: Rabbit polyclonal antibodies used were anti–caveolin-1, anti-TLR2, anti–IRAK-1, anti-MYD88, anti-TRAF6, and anti-TLR4 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA); anti-TLR2 antisera from Tularik (South San Francisco, California, USA); and anti-asialoGM1 (Wako, Richmond, Virginia, USA).

Techniques: Labeling, Infection, Transmission Assay, Flow Cytometry

Journal:

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

doi: 10.1172/JCI200420773

Figure Lengend Snippet: Mobilization of TLR2 and asialoGM1 in response to bacteria or bacterial ligands. Flow cytometry was used to quantify exposed asialoGM1, TLR2, and TLR4 on primary (HNP) cells (A) or 16HBE cells (B) after stimulation with P. aeruginosa or S. aureus or with monoclonal anti-asialoGM1, Pam3Cys-Ser-Lys4 (Pam3Cys), a TLR2 ligand, or LPS, a TLR4 ligand. Peaks outlined with a thin black line indicate binding by secondary antibody alone; gray-shaded peaks represent the labeled population under control conditions; and peaks demarcated by the heavy black line represent the population after stimulation.

Article Snippet: Rabbit polyclonal antibodies used were anti–caveolin-1, anti-TLR2, anti–IRAK-1, anti-MYD88, anti-TRAF6, and anti-TLR4 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA); anti-TLR2 antisera from Tularik (South San Francisco, California, USA); and anti-asialoGM1 (Wako, Richmond, Virginia, USA).

Techniques: Flow Cytometry, Binding Assay, Labeling

Journal:

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

doi: 10.1172/JCI200420773

Figure Lengend Snippet: Activation of NF-κB and IL-8 by bacteria or bacterial agonists is inhibited by DN mutations in TLR2 and MyD88. (A) IL-8 expression in 1HAEo- cells transfected with plasmids containing wild-type, DN TLR2, or a vector control was quantified by ELISA after exposure to P. aeruginosa PAO1, S. aureus RN6390, or anti-asialoGM1. Values represent the fold increase in IL-8 compared with that of unstimulated cells. IL-8 in cells stimulated by PAO1 was 1.9–2.5 ng/ml (*P < 0.001). (B) NF-κB luciferase activity in 1HAEo- cells transfected with plasmids expressing TLR2 DN or MyD88 DN constructs compared with that of cells transfected with the corresponding empty vector control was significantly inhibited after stimulation with P. aeruginosa PAO1, S. aureus RN6390, anti-asialoGM1, or Pam3Cys-Ser-Lys4 (*P < 0.001 for each). The TLR4 DN construct did not inhibit NF-κB luciferase activity. NF-κB luciferase activity for cells expressing the control vector was normalized for each stimulus and represents three- to fivefold increases over that of unstimulated cells. (C) Inhibition of IL-8 activation in the presence of filipin. IL-8 production induced by P. aeruginosa, S. aureus, anti-asialoGM1 (aGM1), or Pam3Cys-Ser-Lys4, but not TNF-α, was significantly reduced by filipin (P < 0.05, P < 0.001, P < 0.05, and P > 0.05, respectively).

Article Snippet: Rabbit polyclonal antibodies used were anti–caveolin-1, anti-TLR2, anti–IRAK-1, anti-MYD88, anti-TRAF6, and anti-TLR4 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA); anti-TLR2 antisera from Tularik (South San Francisco, California, USA); and anti-asialoGM1 (Wako, Richmond, Virginia, USA).

Techniques: Activation Assay, Expressing, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Construct, Inhibition

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

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