Journal: British Journal of Pharmacology

Article Title: TLR9 and MyD88 are crucial for the maturation and activation of dendritic cells by paromomycin–miltefosine combination therapy in visceral leishmaniasis

doi: 10.1111/bph.12530

Figure Lengend Snippet: Interaction of TLR9 with paromomycin and miltefosine. (A) Cartoon diagram showing interactions of TLR9 with drugs; TLR9 interacts with anti-leishmanial drugs, paromomycin (A), miltefosine (B) or both drugs in combination (C). The surface representation of TLR9 receptor is shown in blue, paromomycin in green and miltefosine in red respectively. The interacting residues in the inhibitory residual interface region are further enlarged in the box. Side chains of the amino acids contributing to hydrogen bond formation are represented as a stick model with the residue names and numbers shown next to them. TLR9 side chains stick model atom, which interacts are shown in yellow and atoms are in green. Black dotted lines represent the hydrogen bonds. These pictures were generated by PyMol v0.99. (D,E) Biophysical characterization of paromomycin and miltefosine binding with human TLR9. Different concentrations of paromomycin/neomycin or miltefosine/ilmofosine were coated on elisa plates. Human recombinant TLR9 partial protein (10μg mL−1) was added, and the drug-bound TLR9 was detected by adding biotin-TLR9 monoclonal antibody followed by streptavidin-HRP conjugate substrate. Absorbance with 30 μM paromomycin (in D) and 3 μM miltefosine (in E) was set as 100%.

Article Snippet: After washing with PBST five times, the indicated concentration of biotin-TLR9 MAb (Imgenex, San Diego, CA, USA) was added and incubated at room temperature for 1 h. After 5 washings, streptavidin-coupled HRP conjugate (Thermo Pierce, Rockford, IL, USA) was diluted at the ratio of 1:2000 and added into the wells and incubated at RT for 1 h. After a further 7 washings, 100 μL of TMB substrate reagents (Sigma-Aldrich) were added per well and incubated at RT for 10–30 min; 50 μL of 1 M H 3 PO 4 was subsequently added to each well to stop the colour reaction and the absorbance at 450 nm was measured on a Beckman-Coulter DTX microplate reader (Beckman Coulter, Fullerton, CA, USA).

Techniques: Residue, Generated, Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: British Journal of Pharmacology

Article Title: TLR9 and MyD88 are crucial for the maturation and activation of dendritic cells by paromomycin–miltefosine combination therapy in visceral leishmaniasis

doi: 10.1111/bph.12530

Figure Lengend Snippet: Paromomycin/miltefosine combination induces TLR9-dependent modulation of effector functions in DCs. (A,B) The control DCs (n = 8) were either transfected with control siRNA or TLR9 siRNA and were then infected with Leishmania donovani parasites for 24 h (see Methods). Parasitized DCs were then treated with paromomycin and miltefosine, alone or in combination. RNA was isolated and levels of mRNA expression for IL-12p40 (A) and IL-10 (B), were determined by quantitative RT-PCR. Results are presented as changes (fold) relative to uninfected control DCs. (C,D) Control DCs, either transfected with control siRNA or TLR9 siRNA, were cultured in a 24-well plate (1 × 106 cells·mL−1) were pretreated and infected as described above. The culture supernatants were assayed for the levels of IL-12 (C) and IL-10 (D) in the elisa as described in the Methods section. elisa data are expressed as means of values from triplicate experiments that yielded similar observations. The data represent the mean ± SEM of results from three independent experiments of control subjects (n = 8) that yielded similar results. (E,F) Paromomycin and miltefosine promotes TLR9-mediated effector function in AVL patients: BMDCs of VL patients (n = 10) were stimulated ex vivo with the combination of paromomycin–miltefosine with/without pretreatment with human inhibitory ODN (TLR9 blocker) and the production of IL-12 (E) and IL-10 (F) was estimated at the release level by elisa. The geometric mean of cytokine production of all populations was determined per donor and compared between the stimulated and non-stimulated groups. Horizontal bars denote the median values. Each dot represents one individual.

Article Snippet: After washing with PBST five times, the indicated concentration of biotin-TLR9 MAb (Imgenex, San Diego, CA, USA) was added and incubated at room temperature for 1 h. After 5 washings, streptavidin-coupled HRP conjugate (Thermo Pierce, Rockford, IL, USA) was diluted at the ratio of 1:2000 and added into the wells and incubated at RT for 1 h. After a further 7 washings, 100 μL of TMB substrate reagents (Sigma-Aldrich) were added per well and incubated at RT for 10–30 min; 50 μL of 1 M H 3 PO 4 was subsequently added to each well to stop the colour reaction and the absorbance at 450 nm was measured on a Beckman-Coulter DTX microplate reader (Beckman Coulter, Fullerton, CA, USA).

Techniques: Transfection, Infection, Isolation, Expressing, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Ex Vivo

Journal: British Journal of Pharmacology

Article Title: TLR9 and MyD88 are crucial for the maturation and activation of dendritic cells by paromomycin–miltefosine combination therapy in visceral leishmaniasis

doi: 10.1111/bph.12530

Figure Lengend Snippet: Paromomycin/miltefosine combination interacts with TLR9 in vitro and activates NF-κB promoter activity via TLR9. (A) Kinetics of changes in intracellular TLR9 expression in cultured DCs of control healthy subjects (n = 10) with stimulations by various concentrations (μM) of paromomycin or miltefosine (alone or combined) or equimolar doses of OVA protein (control) for 12 h at 37°C. Here, 1:10 for example means 1 μM of miltefosine and 10 μM of paromomycin used single or in combination, and so on. Results denote differential modulation of TLR9 expressions with respect to stimulations; quantitative flowcytometric data are expressed as mean fluorescence intensity (MFI; arbitrary units). Asterisks denote P < 0.05(Mann Whitney U test). (B,C) Dose–response of paromomycin and miltefosine [alone (B) or combined (C) ] for NF-κB promoter activity. HEK293-TLR9 cells were transfected with NF-κB reporter luciferase plasmid or empty vector (see Methods section). Cells were pre-incubated with or without PMB (10 μg·mL−1) and then stimulated with different doses of paromomycin or miltefosine (1–30 μM; alone or combinational ratios) or 0.1 μg·mL−1 OVA and incubated for 12 h. Then cell extracts were prepared for determination of luciferase activity. Results represent the ratio of luciferase (luc) to β-galactosidase (β-gal) and are the mean ± SEM. of three experiments performed in triplicate. Asterisks denote P < 0.05 versus protein control, OVA (Mann–Whitney U-test). (D) MyD88 is essential for TLR9-mediated induction of NF-κB promoter activity. 293-TLR9 or HEK293-TLR4 cells were transfected with NF-κB reporter luciferase plasmid or with pSV40/LACZ plasmid and co-transfected with MyD88DN or empty vector (see Methods section). Cells stimulated with miltefosine (M) and paromomycin (P) combination (3 and 30 μM respectively), 0.1 mM CpG 2006, 0.01 μg·mL−1 LPS or 0.1 μg·mL−1 OVA and incubated for 12 h, after which extracts were prepared for determination of luciferase activity. Results represent the ratio of luciferase (Luc) to b-galactosidase (β-gal) and are the mean ± SEM of three independent experiments performed in triplicate. An asterisk denotes P < 0.05 versus protein control, OVA (Mann–Whitney U-test).

Article Snippet: After washing with PBST five times, the indicated concentration of biotin-TLR9 MAb (Imgenex, San Diego, CA, USA) was added and incubated at room temperature for 1 h. After 5 washings, streptavidin-coupled HRP conjugate (Thermo Pierce, Rockford, IL, USA) was diluted at the ratio of 1:2000 and added into the wells and incubated at RT for 1 h. After a further 7 washings, 100 μL of TMB substrate reagents (Sigma-Aldrich) were added per well and incubated at RT for 10–30 min; 50 μL of 1 M H 3 PO 4 was subsequently added to each well to stop the colour reaction and the absorbance at 450 nm was measured on a Beckman-Coulter DTX microplate reader (Beckman Coulter, Fullerton, CA, USA).

Techniques: In Vitro, Activity Assay, Expressing, Cell Culture, Fluorescence, MANN-WHITNEY, Transfection, Luciferase, Plasmid Preparation, Incubation

Journal: British Journal of Pharmacology

Article Title: TLR9 and MyD88 are crucial for the maturation and activation of dendritic cells by paromomycin–miltefosine combination therapy in visceral leishmaniasis

doi: 10.1111/bph.12530

Figure Lengend Snippet: TLR9 and MyD88 are required for optimal stimulation of CD4+ T-cells by paromomycin/miltefosine-stimulated DCs in VL patients. Analysis of paromomycin/miltefosine-stimulated and unstimulated BMDCs from VL patients (n = 10). At day 6 of the BMDC culture, cells were pretransfected with/without control siRNA or TLR9 siRNA or TLR4 siRNA or MyD88DN plasmid and later cultured with or without the combination of paromomycin/miltefosine (30 and 3 μM respectively) or 0.01 μg·mL−1 LPS or 0.1 mM CpG 2006 in a fresh medium for 2 more days as described in Methods section. (A) Flow cytometric analysis of paromomycin/miltefosine-stimulated and unstimulated BMDCs from VL patients for CD40 surface expression. Then DCs were transfected for RNA interference, stimulated as above, stained with PE-conjugated CD40 mAb and analysed with FACS. The Western blot gel demonstrates total protein levels of TLR9, TLR4 and MyD88 in control and knock-out sets. (B) Allostimulatory activity of BM DCs from VL patients. To measure the allostimulatory activity of BMDCs, differentially transfected and treated DCs (as above) were irradiated and incubated with allogeneic CD4+ T-cells of the same patient. T-cell responses were evaluated by [3H]-thymidine incorporation of CD4+ T-cells. The data indicate mean ± SEM of triplicate samples of one representative experiment. (C) IFN-γ production from restimulated T-cells was measured by elisa and expressed as pg·mL−1. The data indicate mean ± SEM of triplicate samples of one representative of three independent experiments. An asterisk denotes P < 0.05 versus control (Mann–Whitney U-test).

Article Snippet: After washing with PBST five times, the indicated concentration of biotin-TLR9 MAb (Imgenex, San Diego, CA, USA) was added and incubated at room temperature for 1 h. After 5 washings, streptavidin-coupled HRP conjugate (Thermo Pierce, Rockford, IL, USA) was diluted at the ratio of 1:2000 and added into the wells and incubated at RT for 1 h. After a further 7 washings, 100 μL of TMB substrate reagents (Sigma-Aldrich) were added per well and incubated at RT for 10–30 min; 50 μL of 1 M H 3 PO 4 was subsequently added to each well to stop the colour reaction and the absorbance at 450 nm was measured on a Beckman-Coulter DTX microplate reader (Beckman Coulter, Fullerton, CA, USA).

Techniques: Plasmid Preparation, Cell Culture, Expressing, Transfection, Staining, Western Blot, Knock-Out, Activity Assay, Irradiation, Incubation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: British Journal of Pharmacology

Article Title: TLR9 and MyD88 are crucial for the maturation and activation of dendritic cells by paromomycin–miltefosine combination therapy in visceral leishmaniasis

doi: 10.1111/bph.12530

Figure Lengend Snippet: Phenotypic DC maturation by paromomycin/miltefosine combination is impaired in TLR9-deficient DCs. (A–D) Control monocyte-derived DCs (1 × 106 cells mL-1) were either transfected with/without control siRNA or TLR9 siRNA and were then infected with/without Leishmania donovani parasites for 24 h. Parasitized DCs were then treated with paromomycin and miltefosine alone/or in combination. After 24 h of incubation treated macrophages were analysed by flow cytometry for CD40 or CD80 or CD86 or MHC-II (FL2-H) expression as described in Methods section. Results are representative of at least three independent experiments of different patients (n = 7). An asterisk denotes P < 0.05 versus control (Mann–Whitney U-test).

Article Snippet: After washing with PBST five times, the indicated concentration of biotin-TLR9 MAb (Imgenex, San Diego, CA, USA) was added and incubated at room temperature for 1 h. After 5 washings, streptavidin-coupled HRP conjugate (Thermo Pierce, Rockford, IL, USA) was diluted at the ratio of 1:2000 and added into the wells and incubated at RT for 1 h. After a further 7 washings, 100 μL of TMB substrate reagents (Sigma-Aldrich) were added per well and incubated at RT for 10–30 min; 50 μL of 1 M H 3 PO 4 was subsequently added to each well to stop the colour reaction and the absorbance at 450 nm was measured on a Beckman-Coulter DTX microplate reader (Beckman Coulter, Fullerton, CA, USA).

Techniques: Derivative Assay, Transfection, Infection, Incubation, Flow Cytometry, Expressing, MANN-WHITNEY

Journal: British Journal of Pharmacology

Article Title: TLR9 and MyD88 are crucial for the maturation and activation of dendritic cells by paromomycin–miltefosine combination therapy in visceral leishmaniasis

doi: 10.1111/bph.12530

Figure Lengend Snippet: Role of TLR9 in paromomycin/miltefosine-induced histone H3 modifications at the IL-12 and IL-10 promoter in infected DCs. Control DCs (1 × 106 cells per mL) from healthy volunteers (n = 6) were either transfected with control siRNA or TLR9 siRNA and were then infected with Leishmania donovani parasites for 24 h. Parasitized DCs were then treated with paromomycin and miltefosine; alone or in combination. ChIP assays were conducted as described in the Methods section. Immunoprecipitations were performed using Abs specific to phosphorylated H3 [IP phospho-H3; (A,C) ] or acetylated H3 [IP acetyl-H3; (B,D) ], and conventional RT-PCR or quantitative real-time PCR was performed using primers specific to the IL-12 (A,B,E) or IL-10 (C,D,F) promoter. *P < 0.001 compared with infected DCs without the treatment. (E-F) Changes in phosphorylated/acetylated H3 modifications at IL-12 locus (E) and IL-10 locus (F) with mono treatments of DCs with paromomycin or miltefosine are shown. Fold changes over input DNA are demonstrated.

Article Snippet: After washing with PBST five times, the indicated concentration of biotin-TLR9 MAb (Imgenex, San Diego, CA, USA) was added and incubated at room temperature for 1 h. After 5 washings, streptavidin-coupled HRP conjugate (Thermo Pierce, Rockford, IL, USA) was diluted at the ratio of 1:2000 and added into the wells and incubated at RT for 1 h. After a further 7 washings, 100 μL of TMB substrate reagents (Sigma-Aldrich) were added per well and incubated at RT for 10–30 min; 50 μL of 1 M H 3 PO 4 was subsequently added to each well to stop the colour reaction and the absorbance at 450 nm was measured on a Beckman-Coulter DTX microplate reader (Beckman Coulter, Fullerton, CA, USA).

Techniques: Infection, Transfection, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: British Journal of Pharmacology

Article Title: TLR9 and MyD88 are crucial for the maturation and activation of dendritic cells by paromomycin–miltefosine combination therapy in visceral leishmaniasis

doi: 10.1111/bph.12530

Figure Lengend Snippet: Interaction of TLR9 with paromomycin and miltefosine

Article Snippet: After washing with PBST five times, the indicated concentration of biotin-TLR9 MAb (Imgenex, San Diego, CA, USA) was added and incubated at room temperature for 1 h. After 5 washings, streptavidin-coupled HRP conjugate (Thermo Pierce, Rockford, IL, USA) was diluted at the ratio of 1:2000 and added into the wells and incubated at RT for 1 h. After a further 7 washings, 100 μL of TMB substrate reagents (Sigma-Aldrich) were added per well and incubated at RT for 10–30 min; 50 μL of 1 M H 3 PO 4 was subsequently added to each well to stop the colour reaction and the absorbance at 450 nm was measured on a Beckman-Coulter DTX microplate reader (Beckman Coulter, Fullerton, CA, USA).

Techniques: Binding Assay, Residue