cd14 fc recombinant protein  (R&D Systems)

Journal: PloS one

Article Title: Mycobacterial PIMs inhibit host inflammatory responses through CD14-dependent and CD14-independent mechanisms.

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Figure 4. Synthetic PIM analogues inhibit S-LPS-binding to soluble CD14. The effects of PIMs on the binding of biotinylated S-LPS to sCD14 was investigated in presence of 1% FCS (A, D), or 0.1% serum from wild-type mice (B) or from LBP-deficient mice (C). Solid phase adsorbed sCD14 was incubated (1 hr at 37uC) in the presence of synthetic PI, PIM1, isoPIM1, deAcPIM2 mimetic and PIM2 mimetic (all at 10 mg/mL) or vehicle, before addition of biotinylated S-LPS (0.1 mg/mL; 2 hrs at 37uC). Binding specificity was determined by incubation with increasing concentrations of non biotinylated S-LPS 1 hr prior to biotinylated S-LPS (D). Results are expressed as percentage of biotinylated S-LPS-binding to sCD14 as compared to incubation with vehicle and are mean +/2 SD from three independent experiments. ***, p,0.001 versus vehicle; hhh, p,0.001 indicate significant differences between isoPIM1 versus PI as control; {{{, p,0.001 indicate significant differences between PIM2 mimetic and deAcPIM2 mimetic as control. doi:10.1371/journal.pone.0024631.g004

Article Snippet: LPS binding to soluble CD14 Soluble recombinant mouse CD14 was coated overnight at 4uC (5 mg/mL on Nunc 96-well plates; R&D systems, Abingdon, UK) and non specific binding saturated with 2% BSA in PBS for 1 hr at 37uC.

Techniques: Analogues, Binding Assay, Incubation, Control

Journal: PloS one

Article Title: Mycobacterial PIMs inhibit host inflammatory responses through CD14-dependent and CD14-independent mechanisms.

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Figure 5. Inhibition of TLR2 signaling by PIM analogues is independent of CD14. Macrophages from C57Bl/6 mice (A, C) or CD14 KO mice (B, D) were incubated with synthetic PI, isoPIM1, deAcPIM2 mimetic, PIM2 mimetic (10 mg/mL) or control vehicle prior to stimulation with Malp2 (30 ng/mL; A, B) or Pam3CSK4 (Pam3; 0.5 mg/mL; C, D). TNF release was measured in supernatants after overnight incubation. Results are mean +/2 SD from n = 4 mice from two independent experiments. *, p,0.05; **, p,0.01; ***, p,0.001 versus vehicle. hh, p,0.01; hhh, p,0.001 indicate significant differences between isoPIM1 versus PI as control; {, p,0.05; {{{, p,0.001, indicate significant differences between PIM2 mimetic versus deAcPIM2 mimetic as control. doi:10.1371/journal.pone.0024631.g005

Article Snippet: LPS binding to soluble CD14 Soluble recombinant mouse CD14 was coated overnight at 4uC (5 mg/mL on Nunc 96-well plates; R&D systems, Abingdon, UK) and non specific binding saturated with 2% BSA in PBS for 1 hr at 37uC.

Techniques: Inhibition, Analogues, Incubation, Control

Journal: PloS one

Article Title: Mycobacterial PIMs inhibit host inflammatory responses through CD14-dependent and CD14-independent mechanisms.

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Figure 6. CD14-dependency of TNF and IL-12 p40 release induced by S-LPS versus Re-LPS. Macrophages from C57Bl/6 or CD14 KO mice were stimulated with increasing concentrations of S-LPS (A, B) or Re-LPS (C, D). TNF (A, C) and IL-12 p40 (B, D) concentrations were measured in the supernatants after overnight incubation. Results are mean +/2 SEM from n = 4 mice from two independent experiments. *, p,0.05; **, p,0.01; ***, p,0.001 indicate significant differences between C57Bl/6 and CD14 KO. doi:10.1371/journal.pone.0024631.g006

Article Snippet: LPS binding to soluble CD14 Soluble recombinant mouse CD14 was coated overnight at 4uC (5 mg/mL on Nunc 96-well plates; R&D systems, Abingdon, UK) and non specific binding saturated with 2% BSA in PBS for 1 hr at 37uC.

Techniques: Incubation

Journal: PloS one

Article Title: Mycobacterial PIMs inhibit host inflammatory responses through CD14-dependent and CD14-independent mechanisms.

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Figure 7. Differential inhibition of S-LPS versus Re-LPS induced TNF and IL-12 p40 release by PIMs. Concentrations of TNF (A–C) and IL- 12 p40 (D–F) in supernatants of wild type (A, B, D, E) or CD14-deficient (C, F) macrophages stimulated overnight with 100 ng/mL of S-LPS (A, D) or Re- LPS (B, C, E, F) in the presence of synthetic PI, isoPIM1, deAcPIM2 mimetic, PIM2 mimetic (10 mg/mL), or vehicle. Results are mean +/2 SD from n = 4 mice from two independent experiments representative of three independent experiments. ND: not detected. **, p,0.01; ***, p,0.001 versus vehicle. hh, p,0.01; hhh, p,0.001 indicate significant differences between isoPIM1 versus PI as control; {{, p,0.01; {{{, p,0.001 indicate significant differences between PIM2 mimetic versus deAcPIM2 mimetic as control. doi:10.1371/journal.pone.0024631.g007

Article Snippet: LPS binding to soluble CD14 Soluble recombinant mouse CD14 was coated overnight at 4uC (5 mg/mL on Nunc 96-well plates; R&D systems, Abingdon, UK) and non specific binding saturated with 2% BSA in PBS for 1 hr at 37uC.

Techniques: Inhibition, Control

Journal: PloS one

Article Title: Mycobacterial PIMs inhibit host inflammatory responses through CD14-dependent and CD14-independent mechanisms.

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Figure 4. Synthetic PIM analogues inhibit S-LPS-binding to soluble CD14. The effects of PIMs on the binding of biotinylated S-LPS to sCD14 was investigated in presence of 1% FCS (A, D), or 0.1% serum from wild-type mice (B) or from LBP-deficient mice (C). Solid phase adsorbed sCD14 was incubated (1 hr at 37uC) in the presence of synthetic PI, PIM1, isoPIM1, deAcPIM2 mimetic and PIM2 mimetic (all at 10 mg/mL) or vehicle, before addition of biotinylated S-LPS (0.1 mg/mL; 2 hrs at 37uC). Binding specificity was determined by incubation with increasing concentrations of non biotinylated S-LPS 1 hr prior to biotinylated S-LPS (D). Results are expressed as percentage of biotinylated S-LPS-binding to sCD14 as compared to incubation with vehicle and are mean +/2 SD from three independent experiments. ***, p,0.001 versus vehicle; hhh, p,0.001 indicate significant differences between isoPIM1 versus PI as control; {{{, p,0.001 indicate significant differences between PIM2 mimetic and deAcPIM2 mimetic as control. doi:10.1371/journal.pone.0024631.g004

Article Snippet: PIMs were incubated in presence or absence of 5 mg/mL recombinant mouse CD14 Fc chimera (endotoxin ,1.0EU/mg protein; R&D system).

Techniques: Analogues, Binding Assay, Incubation, Control

Journal: PloS one

Article Title: Mycobacterial PIMs inhibit host inflammatory responses through CD14-dependent and CD14-independent mechanisms.

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Figure 5. Inhibition of TLR2 signaling by PIM analogues is independent of CD14. Macrophages from C57Bl/6 mice (A, C) or CD14 KO mice (B, D) were incubated with synthetic PI, isoPIM1, deAcPIM2 mimetic, PIM2 mimetic (10 mg/mL) or control vehicle prior to stimulation with Malp2 (30 ng/mL; A, B) or Pam3CSK4 (Pam3; 0.5 mg/mL; C, D). TNF release was measured in supernatants after overnight incubation. Results are mean +/2 SD from n = 4 mice from two independent experiments. *, p,0.05; **, p,0.01; ***, p,0.001 versus vehicle. hh, p,0.01; hhh, p,0.001 indicate significant differences between isoPIM1 versus PI as control; {, p,0.05; {{{, p,0.001, indicate significant differences between PIM2 mimetic versus deAcPIM2 mimetic as control. doi:10.1371/journal.pone.0024631.g005

Article Snippet: PIMs were incubated in presence or absence of 5 mg/mL recombinant mouse CD14 Fc chimera (endotoxin ,1.0EU/mg protein; R&D system).

Techniques: Inhibition, Analogues, Incubation, Control

Journal: PloS one

Article Title: Mycobacterial PIMs inhibit host inflammatory responses through CD14-dependent and CD14-independent mechanisms.

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Figure 6. CD14-dependency of TNF and IL-12 p40 release induced by S-LPS versus Re-LPS. Macrophages from C57Bl/6 or CD14 KO mice were stimulated with increasing concentrations of S-LPS (A, B) or Re-LPS (C, D). TNF (A, C) and IL-12 p40 (B, D) concentrations were measured in the supernatants after overnight incubation. Results are mean +/2 SEM from n = 4 mice from two independent experiments. *, p,0.05; **, p,0.01; ***, p,0.001 indicate significant differences between C57Bl/6 and CD14 KO. doi:10.1371/journal.pone.0024631.g006

Article Snippet: PIMs were incubated in presence or absence of 5 mg/mL recombinant mouse CD14 Fc chimera (endotoxin ,1.0EU/mg protein; R&D system).

Techniques: Incubation

Journal: PloS one

Article Title: Mycobacterial PIMs inhibit host inflammatory responses through CD14-dependent and CD14-independent mechanisms.

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Figure 7. Differential inhibition of S-LPS versus Re-LPS induced TNF and IL-12 p40 release by PIMs. Concentrations of TNF (A–C) and IL- 12 p40 (D–F) in supernatants of wild type (A, B, D, E) or CD14-deficient (C, F) macrophages stimulated overnight with 100 ng/mL of S-LPS (A, D) or Re- LPS (B, C, E, F) in the presence of synthetic PI, isoPIM1, deAcPIM2 mimetic, PIM2 mimetic (10 mg/mL), or vehicle. Results are mean +/2 SD from n = 4 mice from two independent experiments representative of three independent experiments. ND: not detected. **, p,0.01; ***, p,0.001 versus vehicle. hh, p,0.01; hhh, p,0.001 indicate significant differences between isoPIM1 versus PI as control; {{, p,0.01; {{{, p,0.001 indicate significant differences between PIM2 mimetic versus deAcPIM2 mimetic as control. doi:10.1371/journal.pone.0024631.g007

Article Snippet: PIMs were incubated in presence or absence of 5 mg/mL recombinant mouse CD14 Fc chimera (endotoxin ,1.0EU/mg protein; R&D system).

Techniques: Inhibition, Control

Journal: PLoS ONE

Article Title: Mycobacterial PIMs Inhibit Host Inflammatory Responses through CD14-Dependent and CD14-Independent Mechanisms

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Bone marrow derived macrophages (A, B) were stimulated with increasing concentrations of S-LPS in presence of 10 µg/mL of PIM 2 mimetic or deAcPIM 2 mimetic, or vehicle control, and TNF (A) and IL-12 p40 (B) were measured in supernatants after overnight incubation. Results are mean +/− SEM from n = 6 mice from three independent experiments. PIM analogues were titrated (C, D) in the presence of 0.1 µg/mL S-LPS and a 10 µg/mL dose was chosen as this concentration was sufficient for the active PIMs to strongly inhibit LPS-induced TNF (C) and IL-12 p40 (D) release without cytotoxicity. (E–J) HEK cells stably transfected with TLR4, MD2 and CD14 were incubated with PI (E), PIM 1 (F), isoPIM 1 (G), deAcPIM 2 mimetic (H) or PIM 2 mimetic (I) (10 µg/mL; dotted line) prior to incubation with biotinylated S-LPS (2,5 µg/mL) and streptavidin FITC (black line) or only streptavidin FITC (grey histogram). Non transfected HEK cells were incubated with biotinylated S-LPS as a control (J). Results are from one experiment representative of three independent experiments. (K) Percentage of S-LPS-binding to HEK-MTC cells in presence of vehicle, PI, isoPIM 1 , deAcPIM 2 mimetic or PIM 2 mimetic. Results are the mean +/− SD from three independent experiments. (L) Human IL-8 was measured in the supernatant after overnight S-LPS stimulation of HEK-MTC cells. Results are mean +/− SD from triplicates, from one experiment representative of three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001 versus vehicle; θ, p<0.05; θθθ, p<0.001 indicate significant differences between PIM 1 or isoPIM 1 versus PI as control; ††, p<0.01; †††, p<0.001 indicate significant differences between PIM 2 mimetic and deAcPIM 2 mimetic as control.

Article Snippet: Soluble recombinant mouse CD14 was coated overnight at 4°C (5 μg/mL on Nunc 96-well plates; R&D systems, Abingdon, UK) and non specific binding saturated with 2% BSA in PBS for 1 hr at 37°C.

Techniques: Derivative Assay, Control, Incubation, Analogues, Concentration Assay, Stable Transfection, Transfection, Binding Assay

Journal: PLoS ONE

Article Title: Mycobacterial PIMs Inhibit Host Inflammatory Responses through CD14-Dependent and CD14-Independent Mechanisms

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Macrophages from C57Bl/6 mice (A, C) or CD14 KO mice (B, D) were incubated with synthetic PI, isoPIM 1 , deAcPIM 2 mimetic, PIM 2 mimetic (10 µg/mL) or control vehicle prior to stimulation with Malp2 (30 ng/mL; A, B) or Pam 3 CSK 4 (Pam 3 ; 0.5 µg/mL; C, D). TNF release was measured in supernatants after overnight incubation. Results are mean +/− SD from n = 4 mice from two independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001 versus vehicle. θθ, p<0.01; θθθ, p<0.001 indicate significant differences between isoPIM 1 versus PI as control; †, p<0.05; †††, p<0.001, indicate significant differences between PIM 2 mimetic versus deAcPIM 2 mimetic as control.

Article Snippet: Soluble recombinant mouse CD14 was coated overnight at 4°C (5 μg/mL on Nunc 96-well plates; R&D systems, Abingdon, UK) and non specific binding saturated with 2% BSA in PBS for 1 hr at 37°C.

Techniques: Incubation, Control

Journal: PLoS ONE

Article Title: Mycobacterial PIMs Inhibit Host Inflammatory Responses through CD14-Dependent and CD14-Independent Mechanisms

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Macrophages from C57Bl/6 or CD14 KO mice were stimulated with increasing concentrations of S-LPS (A, B) or Re-LPS (C, D). TNF (A, C) and IL-12 p40 (B, D) concentrations were measured in the supernatants after overnight incubation. Results are mean +/− SEM from n = 4 mice from two independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001 indicate significant differences between C57Bl/6 and CD14 KO.

Article Snippet: Soluble recombinant mouse CD14 was coated overnight at 4°C (5 μg/mL on Nunc 96-well plates; R&D systems, Abingdon, UK) and non specific binding saturated with 2% BSA in PBS for 1 hr at 37°C.

Techniques: Incubation

Journal: PLoS ONE

Article Title: Mycobacterial PIMs Inhibit Host Inflammatory Responses through CD14-Dependent and CD14-Independent Mechanisms

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Concentrations of TNF (A–C) and IL-12 p40 (D–F) in supernatants of wild type (A, B, D, E) or CD14-deficient (C, F) macrophages stimulated overnight with 100 ng/mL of S-LPS (A, D) or Re-LPS (B, C, E, F) in the presence of synthetic PI, isoPIM 1 , deAcPIM 2 mimetic, PIM 2 mimetic (10 µg/mL), or vehicle. Results are mean +/− SD from n = 4 mice from two independent experiments representative of three independent experiments. ND: not detected. **, p<0.01; ***, p<0.001 versus vehicle. θθ, p<0.01; θθθ, p<0.001 indicate significant differences between isoPIM 1 versus PI as control; ††, p<0.01; †††, p<0.001 indicate significant differences between PIM 2 mimetic versus deAcPIM 2 mimetic as control.

Article Snippet: Soluble recombinant mouse CD14 was coated overnight at 4°C (5 μg/mL on Nunc 96-well plates; R&D systems, Abingdon, UK) and non specific binding saturated with 2% BSA in PBS for 1 hr at 37°C.

Techniques: Control