Journal: PloS one

Article Title: Immunogenecity of modified alkane polymers is mediated through TLR1/2 activation.

doi: 10.1371/journal.pone.0002438

Figure Lengend Snippet: Figure 5. Modified alkane polymers induce activation of TLR-1 and TLR-2 signaling pathways. a) Luciferase activity expressed by human TLR1/2, TLR2, TLR3 and TLR4 stable HEK 3T3 transfectant (pNF-kB-LUC Stratagene). Cells were left untreated or treated for a different time period with 50 mg/ml of unPE, mPE or pre and post implant PE and respective positive controls; PGN(10 mg/ml) for TLR1/2 and TLR2, Poly (I:C)(1 mg/ml) for TLR3 and LPS (10 mg/ml) for TLR4. doi:10.1371/journal.pone.0002438.g005

Article Snippet: Similar experiments were conducted in presence of mouse anti human TLR2 mAb (clone 383936 R&D Systems) which is known to prevent ligand access to the TLR2 binding groove.

Techniques: Modification, Activation Assay, Protein-Protein interactions, Luciferase, Activity Assay, Transfection

Journal: PloS one

Article Title: Immunogenecity of modified alkane polymers is mediated through TLR1/2 activation.

doi: 10.1371/journal.pone.0002438

Figure Lengend Snippet: Figure 6. Direct binding of oxidized alkane polymers to soluble TLR-2 molecules. a) Left panels; fluorescence emission scans collected for free soluble TLR2 and TLR2 in complex with two different concentrations (exponential and plateau) of each analyzed polymer. Central panels; normalized fluorescence data (DF) for each concentration point as a function of the free ligand concentration. Right panels; fluorescence emission scans collected for free soluble TLR2 and TLR2 in complex with each analyzed polymer at two different concentrations (exponential and plateau) in presence of an anti TLR2 mAb known to block the TLR2 binding groove (stoichiometric ratio 2:1 Ab to soluble TLR2 receptor). b) Comparison of the fluorescence emission scans collected for soluble TLR2, mPE and unPE. doi:10.1371/journal.pone.0002438.g006

Article Snippet: Similar experiments were conducted in presence of mouse anti human TLR2 mAb (clone 383936 R&D Systems) which is known to prevent ligand access to the TLR2 binding groove.

Techniques: Binding Assay, Fluorescence, Polymer, Concentration Assay, Blocking Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Characterization of a Cutibacterium acnes Camp Factor 1-Related Peptide as a New TLR-2 Modulator in In Vitro and Ex Vivo Models of Inflammation

doi: 10.3390/ijms23095065

Figure Lengend Snippet: Expression of Christie–Atkins–Munch–Petersen (CAMP)1 in Cutibacterium acnes ( C. acnes ) strains. C. acnes surface proteins were extracted from a five-day culture ( A , C ) in liquid RCM medium or ( B ) on solid RCM medium and separated by electrophoresis in a 4–12% NuPAGE SDS BisTris gel (50 μg). Separated proteins were transferred onto nitrocellulose membranes, which were incubated ( A , B ) with antibodies against CAMP1 or ( C ) with recombinant toll like receptor (TLR)-2. TLR-2 binding activity was detected with specific biotinylated antibodies against TLR-2, as described in the Materials and Methods. TLR-2 binding activity previously reported by Lheure et al. . Lanes 1 to 12 correspond to phylotype IA1 strains: 75150, 16351, 17248, 53468, 41103, 6919, 78910, 36862, RON, 22197, LIE and 31430, respectively. Lane 13 corresponds to phylotype IA2 strain: CHR. Lanes 14 to 22 correspond to phylotype IB strains: 14230, 12513, 22795, 47474, 27387, 25236, GUE, PIE and 54137, respectively. Lane 23 corresponds to phylotype II strain: 27647. ( D ) HaCaT cells were stimulated for 1, 4, 6 and 24 h at 37 °C, with C. acnes strains 6919 (phylotype IA1), 14230 (phylotype IB) or PIE (phylotype IB) grown in liquid or on solid medium. C-X-C motif chemokine ligand interleukin (CXCL)8/(IL)-8 protein levels were assessed by enzyme-like immunosorbent assay (ELISA). nd: non-determined.

Article Snippet: Unbound antibodies were removed by washing, as described above, and slides were incubated with biotinylated human antibodies against TLR-2 (R&D Systems, Abingdon, UK) diluted to 0.05 μg/mL in TBS/T supplemented with 5% BSA, for 20 h at 4 °C, with gentle mixing.

Techniques: Expressing, Electrophoresis, Incubation, Recombinant, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Characterization of a Cutibacterium acnes Camp Factor 1-Related Peptide as a New TLR-2 Modulator in In Vitro and Ex Vivo Models of Inflammation

doi: 10.3390/ijms23095065

Figure Lengend Snippet: Inhibition of C. acnes -induced CXCL8/IL-8 production by TLR-2 and CAMP1 antibodies. HaCaT cells were treated with ( A ) anti-TLR-2 antibody (1 μg/mL) or ( B ) anti-CAMP1 antibody (1 μg/mL) for 24 h (gray bar), and were then stimulated with various strains of C. acnes (multiplicy of infection 15 (MOI 15) (phylotype IA1, strain 6919; phylotype IB, strains PIE and 14230) for 18 h (dark bar). Control experiments correspond to C. acnes stimulation of HaCaT cells only (gray bar). CXCL8/IL-8 production was measured by ELISA. Statistical significance is indicated by ** ( p < 0.01), *** ( p < 0.001) and **** ( p < 0.0001).

Article Snippet: Unbound antibodies were removed by washing, as described above, and slides were incubated with biotinylated human antibodies against TLR-2 (R&D Systems, Abingdon, UK) diluted to 0.05 μg/mL in TBS/T supplemented with 5% BSA, for 20 h at 4 °C, with gentle mixing.

Techniques: Inhibition, Infection, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Characterization of a Cutibacterium acnes Camp Factor 1-Related Peptide as a New TLR-2 Modulator in In Vitro and Ex Vivo Models of Inflammation

doi: 10.3390/ijms23095065

Figure Lengend Snippet: Specific recognition of C. acnes recombinant CAMP1 by TLR-2. ( A ) The recombinant proteins nmrCAMP1 (nm) and mrCAMP1 (m) (50 μg) were separated by electrophoresis in a 4–12% NuPAGE SDS BisTris gel and transferred onto nitrocellulose membranes, which were incubated with recombinant TLR-2, TLR-1, TLR-4 and TLR-6 (0.1 μg/mL). TLR binding activity was detected with specific biotinylated antibodies against TLR-2, TLR-1, TLR-4 and TLR-6, respectively, as described in the Materials and Methods. The arrow indicates the position of the 47 kDa band of interest. ( B ) The recombinant proteins were separated by electrophoresis in a 4–12% NuPAGE SDS BisTris gel (50 μg) with detection by Coomassie brilliant blue staining. The separated proteins were transferred onto nitrocellulose membranes, which were incubated with poly-histidine antibody or recombinant TLR-2 (0.1 μg/mL), with the detection of TLR binding activity with specific biotinylated antibodies against TLR-2. Lanes 1a and 1b contain the molecular mass markers. Arrows indicate the positions of the 30 and 47 kDa bands of interest. The peptide sequences of the 47 kDa nmrCAMP1 and mrCAMP1 and the 30 kDa protein were obtained by LC-MS/MS (in bold, the amino acid substitutions in mrCAMP1). ( C ) Immobilized nmrCAMP1 (square) and mrCAMP1 (circle) (20 μg/mL) were probed with various concentrations of TLR-1, TLR-2, TLR-4 or TLR-6 (0.016 to 16 μg/mL).

Article Snippet: Unbound antibodies were removed by washing, as described above, and slides were incubated with biotinylated human antibodies against TLR-2 (R&D Systems, Abingdon, UK) diluted to 0.05 μg/mL in TBS/T supplemented with 5% BSA, for 20 h at 4 °C, with gentle mixing.

Techniques: Recombinant, Electrophoresis, Incubation, Binding Assay, Activity Assay, Staining, Liquid Chromatography with Mass Spectroscopy

Journal: International Journal of Molecular Sciences

Article Title: Characterization of a Cutibacterium acnes Camp Factor 1-Related Peptide as a New TLR-2 Modulator in In Vitro and Ex Vivo Models of Inflammation

doi: 10.3390/ijms23095065

Figure Lengend Snippet: Subpeptides from CAMP1 recognized by TLR-2. ( A , B , C ) The peptides derived from CAMP1 proteins: group F (positions A1-C21), group A (positions C22-E9), group C (positions E10-E13), group D (positions E14-E17), group E (positions E18-E20); a biotinylated peptide used as a positive control (position F22) and a peptide used as a negative control (position F24), were immobilized on glass plates. ( A ) The plates were incubated with TLR-2 (10 μg/mL)/anti-TLR-2 antibody (0.1 μg/mL)/HRP (0.5 μg/mL) and ( B ) with anti-TLR-2 antibody (0.1 μg/mL)/HRP (0.5 µg/mL). The arrows indicate the positions of peptides specifically recognized by TLR-2. ( D ) Immobilized rCAMP1 (20 μg/mL) proteins were probed with biotinylated TRL-2 (0.5 μg/mL) (dark bar) after pre-treatment with peptide (A14, A15, B1, B2, C1 and C3) at various concentrations (1.25, 12.5, 125, 1250 and 12,500 nM). Statistical significance is indicated by * ( p < 0.05), ** ( p < 0.01) and *** ( p < 0.001).

Article Snippet: Unbound antibodies were removed by washing, as described above, and slides were incubated with biotinylated human antibodies against TLR-2 (R&D Systems, Abingdon, UK) diluted to 0.05 μg/mL in TBS/T supplemented with 5% BSA, for 20 h at 4 °C, with gentle mixing.

Techniques: Derivative Assay, Positive Control, Negative Control, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Characterization of a Cutibacterium acnes Camp Factor 1-Related Peptide as a New TLR-2 Modulator in In Vitro and Ex Vivo Models of Inflammation

doi: 10.3390/ijms23095065

Figure Lengend Snippet: In silico 3D analysis of C. acnes factor CAMP1. ( A ) Sequence alignment of CAMP factors from different species. Conservatively substituted residues are boxed and strictly conserved residues are highlighted with a red background. The figure was constructed with ESPript 3.0. Reference sequences: Cutibacterium acnes nmCAMP1: AAS92206.1; Cutibacterium acnes mCAMP1: KX581410; Streptococcus agalactiae : ZP_08649639.1; Mobiluncus curtisii : YP_003718285.1; Streptococcus pyogenes : WP_111681137.1. ( B – D ) Predicted structures of the nmCAMP1 and mCAMP1 proteins obtained by homology modeling with the trRosetta server and the images generated with PyMOL software. ( B ) Cartoon representation of CAMP1. The NTD domain is colored in yellow, the CTD domain in cyan and the linker region in red. ( C ) CAMP1 peptides involved in TLR-2 binding are highlighted: A14 in blue, A15 in cyan, B1 in yellow, B2 in orange, B3 in red, C1 in purple and C3 in pink. ( D ) Surface representation of CAMP1 peptides.

Article Snippet: Unbound antibodies were removed by washing, as described above, and slides were incubated with biotinylated human antibodies against TLR-2 (R&D Systems, Abingdon, UK) diluted to 0.05 μg/mL in TBS/T supplemented with 5% BSA, for 20 h at 4 °C, with gentle mixing.

Techniques: In Silico, Sequencing, Construct, Generated, Software, Binding Assay

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

Article Title: Pulmonary surfactant protein A regulates TLR expression and activity in human macrophages.

doi: 10.4049/jimmunol.180.12.7847

Figure Lengend Snippet: FIGURE 1. Human monocytes and macrophages express TLR2 and TLR4. PBMC were isolated from human blood and incubated in Teflon wells with autologous serum. The monocytes differentiate into macrophages (MDM) by day 5. One (A and E)-, 3 (B and F)-, or 5 (C and G)-day-old PBMCs were harvested and incubated with either an allophycocyanin-conjugated human TLR2 mAb (or an allophycocyanin-conjugated subtype control mAb) or a PE-conjugated human TLR4 mAb (or a PE-conjugated subtype control mAb). The stained cells were analyzed using flow cytometry by gating on the monocytes or macrophages (4), and a representative experiment is shown. The number in the top right corner represents the specific MFI (TLR2 or TLR4 MFI subtype control MFI). D and H, Bar graph with cumulative data (triplicate samples in each experiment; n 5 for TLR2; n 4 for TLR4). One-way ANOVA with post-Tukey test, , p 0.005 compared with day 1 cells (SEM).

Article Snippet: Unconjugated mouse anti-human TLR2 (clone TL2.1; Novus Biologicals), mouse anti-human TLR4 (clone HTA125; Gene Tex), mouse IgG2a (clone 20102; R&D Systems), mouse anti-human MR (clone 19.2; BD Biosciences), and mouse IgG1 (R&D Systems) were used for confocal microscopy experiments in which Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes and Invitrogen Detection Technologies) was used as a secondary Ab.

Techniques: Isolation, Incubation, Control, Staining, Cytometry

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

Article Title: Pulmonary surfactant protein A regulates TLR expression and activity in human macrophages.

doi: 10.4049/jimmunol.180.12.7847

Figure Lengend Snippet: FIGURE 2. TLR2 steady-state mRNA levels decrease, while TLR4 levels vary to only a small extent, as monocytes differentiate into macrophages. One-, 3-, and 5- day-old PBMC in Teflon wells were harvested and adhered to a 12-well tissue culture plate in RPMI containing 10% autologous serum. After washing away lymphocytes, the monocytes or MDMs were lysed in TRIzol and total RNA was isolated. mRNA was converted to cDNA and real-time PCR was performed. TLR2 (A), TLR4 (B), and MR (C) amplification was normalized to the -actin housekeeping gene and the RCN number was determined. A and B, Representative experiments (mean SD, triplicate samples) and C is cumulative data (mean SEM) (n 6 (TLR2, TLR4); n 4 (MR)). The MR was used as a positive control as a macrophage marker. One-way ANOVA with post-Tukey test. , p 0.05 compared with day 1 monocytes. , p 0.005 compared with day 1 monocytes.

Article Snippet: Unconjugated mouse anti-human TLR2 (clone TL2.1; Novus Biologicals), mouse anti-human TLR4 (clone HTA125; Gene Tex), mouse IgG2a (clone 20102; R&D Systems), mouse anti-human MR (clone 19.2; BD Biosciences), and mouse IgG1 (R&D Systems) were used for confocal microscopy experiments in which Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes and Invitrogen Detection Technologies) was used as a secondary Ab.

Techniques: Isolation, Real-time Polymerase Chain Reaction, Positive Control, Marker

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

Article Title: Pulmonary surfactant protein A regulates TLR expression and activity in human macrophages.

doi: 10.4049/jimmunol.180.12.7847

Figure Lengend Snippet: FIGURE 3. SP-A increases TLR2, but not TLR4, surface expression on MDMs. Five-day old PBMC in Teflon wells were incubated with or without SP-A (10 g/ml) for 2 h (A and B). Following the same protocol as in Fig. 1, SP-A-treated and untreated MDMs were incubated with either a human allophycocyanin-conjugated TLR2 mAb or allophycocyanin-conjugated subtype control mAb (A) or human PE-conjugated TLR4 mAb or PE-conjugated subtype control mAb (B). The stained cells were analyzed using flow cytometry by gating on the MDMs and the average of triplicate samples is shown in this experiment which is representative of n 4 (A) and n 5 (B). Five-day-old MDMs in monolayer culture on glass coverslips were incubated with or without SP-A (10 g/ml) for 2 h. After washing, cells were fixed in paraformaldehyde (no permeabilization) and stained with mouse anti-human TLR2 mAb, mouse anti-human TLR4 mAb, mouse anti-human MR mAb, or subtype control mAb followed by a secondary Alexa Fluor 488-conjugated anti-mouse Ab. Glass coverslips were mounted and visualized by confocal microscopy. A representative experiment is shown in C (n 3 (TLR2); n 2 (TLR4, MR)). Cumulative data are shown in D. A Student t test was performed. , p 0.05 relative to TLR2 expression on untreated MDMs. The MR was used as a positive control.

Article Snippet: Unconjugated mouse anti-human TLR2 (clone TL2.1; Novus Biologicals), mouse anti-human TLR4 (clone HTA125; Gene Tex), mouse IgG2a (clone 20102; R&D Systems), mouse anti-human MR (clone 19.2; BD Biosciences), and mouse IgG1 (R&D Systems) were used for confocal microscopy experiments in which Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes and Invitrogen Detection Technologies) was used as a secondary Ab.

Techniques: Expressing, Incubation, Control, Staining, Cytometry, Confocal Microscopy, Positive Control

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

Article Title: Pulmonary surfactant protein A regulates TLR expression and activity in human macrophages.

doi: 10.4049/jimmunol.180.12.7847

Figure Lengend Snippet: FIGURE 4. SP-A regulates steady-state mRNA ex- pression of TLR2, but not TLR4, during monocyte dif- ferentiation into macrophages. SP-A (10 g/ml) was added to 1-, 3-, and 5-day-old PBMC in selected Teflon wells for 1, 2, 6, or 20 h. PBMC were harvested and monocytes/macrophages adhered to a 12-well tissue culture plate in autologous serum. After washing away lymphocytes, the monocytes or MDMs were lysed in TRIzol and total RNA was isolated. mRNA was con- verted to cDNA and real-time PCR was performed. TLR2 and TLR4 mRNA amplification was normalized to the -actin housekeeping gene. The fold change was determined by comparing SP-A-treated samples to sam- ples with no SP-A. Cumulative data are shown (SEM) (TLR2: n 6 (day 5, 20 h); n 5 (day 5, 2 h); n 4 (day 1, 1, 2, 6, and 20 h; day 3, 1 and 2 h; day 5 1 and 6 h); n 2 (day 3, 6 and 20 h)) (TLR4: n 6 (day 1, 2 h); n 5 (day 1, 1 and 20 h); n 4 (day 1, 6 h); n 3 (day 3, 1 and 2 h; day 5, 2 and 20 h); n 2 (day 3, 6 h; day 5, 6 h); n 1 (day 3, 20 h; day 5, 20 h)). Student t test was performed. , p 0.05 compared with day 1 monocytes.

Article Snippet: Unconjugated mouse anti-human TLR2 (clone TL2.1; Novus Biologicals), mouse anti-human TLR4 (clone HTA125; Gene Tex), mouse IgG2a (clone 20102; R&D Systems), mouse anti-human MR (clone 19.2; BD Biosciences), and mouse IgG1 (R&D Systems) were used for confocal microscopy experiments in which Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes and Invitrogen Detection Technologies) was used as a secondary Ab.

Techniques: Isolation, Real-time Polymerase Chain Reaction

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

Article Title: Pulmonary surfactant protein A regulates TLR expression and activity in human macrophages.

doi: 10.4049/jimmunol.180.12.7847

Figure Lengend Snippet: FIGURE 8. SP-A is a key regulator of TLR expression and signaling in macrophages. SP-A binds to its receptor(s) leading to increased expression of TLR2 but not TLR4 (this report), the MR () (4) and SR-A () (5). Simultaneously, SP-A regulates TLR activity in response to agonists by decreasing the phosphorylation of key TLR signaling proteins, including Akt and MAPKs. Finally, SP-A decreases the phosphorylation of IB and nuclear translocation of p65 which results in diminished proinflammatory cytokine production.

Article Snippet: Unconjugated mouse anti-human TLR2 (clone TL2.1; Novus Biologicals), mouse anti-human TLR4 (clone HTA125; Gene Tex), mouse IgG2a (clone 20102; R&D Systems), mouse anti-human MR (clone 19.2; BD Biosciences), and mouse IgG1 (R&D Systems) were used for confocal microscopy experiments in which Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes and Invitrogen Detection Technologies) was used as a secondary Ab.

Techniques: Expressing, Activity Assay, Phospho-proteomics, Translocation Assay