Journal: Rheumatology (Oxford, England)

Article Title: Dendritic cells drive profibrotic inflammation and aberrant T cell polarization in systemic sclerosis

doi: 10.1093/rheumatology/keac489

Figure Lengend Snippet: Cytokines produced by TLR-stimulated Mo-DCs from SSc patients. ( A ) The levels of 16 cytokines were measured in the supernatants of monocyte-derived dendritic cells (Mo-DCs) from SSc patients ( n = 38) and healthy controls (HC, n = 10) after exposure to TLR3, TLR4 and TLR9 agonists. Graphs display medians with interquartile range. Significance determined by Mann–Whitney U -test. ( B ) Levels of each cytokine produced by Mo-DCs from patients with SSc in response to TLR3/TLR4/TLR9 stimulation were normalized to median supernatant levels of each cytokine in HCs. Bars show medians with 95% CIs. Significant differences against the controls are coloured orange. ( C ) The expression of CD86 in Mo-DCs from HC and SSc patients was characterized using flow cytometry after TLR stimulation. ( D ) Absolute percentages of Mo-DCs expressing the costimulatory molecule CD86. ( E ) Percentages of Mo-DCs expressing HLA-DR. Mo-DCs were defined as CD14 + CD209 + gated on large cells. Graphs display medians with interquartile ranges. Significance determined by two-way ANOVA. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.001, ns: non-significant

Article Snippet: Mo-DCs were treated with TLR3 ligand Poly(I: C) 10 µg/ml (InvivoGen, San Diego, CA, USA), TLR4 ligand LPS 0.1 µg/ml (InvivoGen), and TLR9 ligand CpG ODN class B 5 µM (InvivoGen).

Techniques: Produced, Derivative Assay, MANN-WHITNEY, Expressing, Flow Cytometry

Journal: Rheumatology (Oxford, England)

Article Title: Dendritic cells drive profibrotic inflammation and aberrant T cell polarization in systemic sclerosis

doi: 10.1093/rheumatology/keac489

Figure Lengend Snippet: T cell polarization patterns induced by TLR-stimulated Mo-DCs from patients with SSc. Peripheral blood mononuclear cells (PBMCs) from patients with systemic sclerosis (SSc, n = 38) and healthy controls (HC, n = 10) were cultured with autologous monocyte-derived dendritic cells (Mo-DCs) stimulated with different TLR agonists. At given times of culture, the percentages of Treg (CD3 + CD4 + CD25 + FoxP3 + ), Th1 (CD3 + CD4 + IFN-γ + ), Th2 (CD3 + CD4 + IL-4 + ), Th17 (CD3 + CD4 + IL-17 + ) and Th22 (CD3 + CD4 + IL-22 + ) cells from total T cells were determined by flow cytometry. ( A ) T cell differentiation patterns induced by Mo-DCs activated with TLR3, TLR4 and TLR9 agonists. Left panels show representative flow cytometry plots. Middle panels show median percentages of each T cell populations. Right panels show medians with interquartile range. Significance determined by Mann–Whitney U -test. ( B ) t -Distributed stochastic neighbour embedding (t-SNE) analysis of CD4 + T cells. The cells positive for each marker or pair of cytokines are displayed in blue. ( C ) Percentages of CD4 + T cells from total T cells expressing the memory marker CD45RO. ( D ) Proportions of dual IFN-γ–IL-4, IFN-γ–IL-17A, IL-4–IL-17A and IL-17A–IL-22 CD4 + T cells from total T cells after exposure to autologous TLR-stimulated Mo-DCs. Graphs show medians with interquartile range. Significance determined by Mann–Whitney U -test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ns: non-significant

Article Snippet: Mo-DCs were treated with TLR3 ligand Poly(I: C) 10 µg/ml (InvivoGen, San Diego, CA, USA), TLR4 ligand LPS 0.1 µg/ml (InvivoGen), and TLR9 ligand CpG ODN class B 5 µM (InvivoGen).

Techniques: Cell Culture, Derivative Assay, Flow Cytometry, Cell Differentiation, MANN-WHITNEY, Marker, Expressing

Journal: Rheumatology (Oxford, England)

Article Title: Dendritic cells drive profibrotic inflammation and aberrant T cell polarization in systemic sclerosis

doi: 10.1093/rheumatology/keac489

Figure Lengend Snippet: Cytokine production by Mo-DCs from patients with lcSSc and dcSSc. ( A ) The levels of 16 cytokines were measured in the supernatants of monocyte-derived dendritic cells (Mo-DCs) from patients with limited cutaneous systemic sclerosis (lcSSc, n = 19), diffuse cutaneous systemic sclerosis (dcSSc, n = 19) and healthy controls (HC, n = 10) after exposure to TLR3, TLR4 and TLR9 agonists. Graphs display medians with interquartile range. Significance determined by Mann–Whitney U -test. * P ≤ 0.05, ** P ≤ 0.02, *** P ≤ 0.001, **** P ≤ 0.0001. ( B ) Levels of each cytokine produced by Mo-DCs from patients with lcSSc and dcSSc in response to TLR3/TLR4/TLR9 stimulation were normalized to median supernatant levels of each cytokine in HCs. Bars show medians with 95% CIs

Article Snippet: Mo-DCs were treated with TLR3 ligand Poly(I: C) 10 µg/ml (InvivoGen, San Diego, CA, USA), TLR4 ligand LPS 0.1 µg/ml (InvivoGen), and TLR9 ligand CpG ODN class B 5 µM (InvivoGen).

Techniques: Derivative Assay, MANN-WHITNEY, Produced

Journal: Rheumatology (Oxford, England)

Article Title: Dendritic cells drive profibrotic inflammation and aberrant T cell polarization in systemic sclerosis

doi: 10.1093/rheumatology/keac489

Figure Lengend Snippet: T cell phenotypes after exposure to TLR-stimulated Mo-DCs from lcSSc and dcSSc patients. ( A ) Peripheral blood mononuclear cells (PBMCs) from limited cutaneous systemic sclerosis (lcSSc, n = 19) and diffuse cutaneous systemic sclerosis SSc (dcSSc, n = 19) patients and healthy controls (HC, n = 10) were cultured with autologous monocyte-derived dendritic cells (Mo-DCs) stimulated with TLR3, TLR4 and TLR9 agonists. At given times of culture, the percentages of Treg, Th1, Th2, Th17 and Th22 cells from total T cells were determined by flow cytometry. Left panels show medians with interquartile ranges. Kruskal–Wallis/Dunn test. Right panels show median percentages of each T cell populations. ( B ) Percentages of dual IFN-γ–IL-4, IFN-γ–IL-17A, IL-4–IL-17A and IL-17A–IL-22 induced by TLR-activated Mo-DCs. * P ≤ 0.05 (dcSSc vs HC), + P ≤ 0.05 (dcSSc vs lcSSc)

Article Snippet: Mo-DCs were treated with TLR3 ligand Poly(I: C) 10 µg/ml (InvivoGen, San Diego, CA, USA), TLR4 ligand LPS 0.1 µg/ml (InvivoGen), and TLR9 ligand CpG ODN class B 5 µM (InvivoGen).

Techniques: Cell Culture, Derivative Assay, Flow Cytometry

Journal: Rheumatology (Oxford, England)

Article Title: Dendritic cells drive profibrotic inflammation and aberrant T cell polarization in systemic sclerosis

doi: 10.1093/rheumatology/keac489

Figure Lengend Snippet: Cytokine production by Mo-DCs from patients with early and late SSc. ( A ) The levels of 16 cytokines were measured in the supernatants of monocyte-derived dendritic cells (Mo-DCs) from patients with early systemic sclerosis (E-SSc, n = 14), late SSc (L-SSc, n = 24) and healthy controls (HC, n = 10) after exposure to TLR3, TLR4 and TLR9 agonists. Graphs display medians with interquartile range. Significance determined by Mann–Whitney U -test. * P ≤ 0.05, ** P ≤ 0.02, *** P ≤ 0.001, **** P ≤ 0.0001. ( B ) Levels of each cytokine produced by Mo-DCs from patients with E-SSc and L-SSc in response to TLR3/TLR4/TLR9 stimulation were normalized to median supernatant levels of each cytokine in HCs. Bars show medians with 95% CIs

Article Snippet: Mo-DCs were treated with TLR3 ligand Poly(I: C) 10 µg/ml (InvivoGen, San Diego, CA, USA), TLR4 ligand LPS 0.1 µg/ml (InvivoGen), and TLR9 ligand CpG ODN class B 5 µM (InvivoGen).

Techniques: Derivative Assay, MANN-WHITNEY, Produced

Journal: Rheumatology (Oxford, England)

Article Title: Dendritic cells drive profibrotic inflammation and aberrant T cell polarization in systemic sclerosis

doi: 10.1093/rheumatology/keac489

Figure Lengend Snippet: T cell phenotypes after exposure to TLR-stimulated Mo-DCs from early and late SSc patients. ( A ) Peripheral blood mononuclear cells (PBMCs) from early systemic sclerosis (E-SSc, n = 14) and late SSc (L-SSc, n = 24) patients, and healthy controls (HC, n = 10) were cultured with autologous monocyte-derived dendritic cells (Mo-DCs) stimulated with TLR3, TLR4 and TLR9 agonists. At given times of culture, the percentages of Treg, Th1, Th2, Th17 and Th22 cells from total T cells were determined by flow cytometry. T cell differentiation patterns induced by TLR3/TLR4/TLR9-activated Mo-DCs. Left panels show medians with interquartile range. Kruskal–Wallis/Dunn test. Middle panels show median percentages of each T cell populations. Right panels show representative flow cytometry dot plots of Th2 cells. ( B ) Percentages of dual IFN-γ–IL-4, IFN-γ–IL-17A, IL-4–IL-17A and IL-17A–IL-22 induced by TLR-activated Mo-DCs. * P ≤ 0.05, **** P ≤ 0.0001 (E-SSc vs HC), ++ P ≤ 0.01, +++ P ≤ 0.001, ++++ P ≤ 0.0001 (E-SSc vs L-SSc)

Article Snippet: Mo-DCs were treated with TLR3 ligand Poly(I: C) 10 µg/ml (InvivoGen, San Diego, CA, USA), TLR4 ligand LPS 0.1 µg/ml (InvivoGen), and TLR9 ligand CpG ODN class B 5 µM (InvivoGen).

Techniques: Cell Culture, Derivative Assay, Flow Cytometry, Cell Differentiation

Journal: PLoS ONE

Article Title: Mitogen-Induced B-Cell Proliferation Activates Chk2-Dependent G1/S Cell Cycle Arrest

doi: 10.1371/journal.pone.0087299

Figure Lengend Snippet: Kinetics of B cell proliferation in response to (A) Epstein-Barr virus infection, (B) constant stimulation with TLR9 ligand CpG (2.5 µg/mL), or (C) CD40L (5 ng/mL) coupled with interleukin-4 (20 pg/mL) treatment. The mean division number based on precursor cohort analysis and plotted over time post infection or treatment is shown. Vertical dashed lines indicate the period of hyper proliferation. (D) The mean division number and time to first division was calculated as in , for the period of hyper-proliferation.

Article Snippet: Thioester stabilized TLR9 ligand CpG ODN 2006 oligonucleotide was purchased from IDT and used at 2.5 µg/ml. mAb G28-5 that binds and activates human CD40 was prepared from a hybridoma cell line (ATCC HB-9110, kind gift of E. Kieff, Harvard Medical School) and used at the final concentration of 1 µg/ml.

Techniques: Infection

Journal: Microorganisms

Article Title: A Journey into Animal Models of Human Osteomyelitis: A Review

doi: 10.3390/microorganisms10061135

Figure Lengend Snippet: Rat models of orthopedic infections.

Article Snippet: Sprague–Dawley , M , 12 w , S. aureus DSM 28763 (field strain isolated from wound infection; genome sequenced, biofilm producer). 10 3 CFU , Implant–related infection , Tibia , na , 42 days , To determine if the prophylactic administration of TLR9 ligand CpG ODN type B would affect a model of implant–related chronic infection , Results indicated that the bacterial load in the infected tibia was reduced at the beginning of infection but failed to prevent the development of chronic infection. , [ ] .

Techniques: Concentration Assay, Isolation, Injection, Produced, Infection, Plasmid Preparation, Activity Assay, Animal Model, Molecular Weight

Journal: Current Research in Virological Science

Article Title: The severe acute respiratory syndrome coronavirus 2 non-structural proteins 1 and 15 proteins mediate antiviral immune evasion

doi: 10.1016/j.crviro.2022.100021

Figure Lengend Snippet: TLR expression in NSP1 and NSP15 stably expressing A549 ​cells. Representative overlapping FACS histogram demonstrating the expression of (A) TLR2 (B) TLR3 (C) TLR4 and (D) TLR9 gated on live (7AAD-) cells to analyze (E) the number of live cells expressing TLR2, TLR3, TLR4 and TLR9 in NSP1 and NSP15 stably expressing A549 ​cells lines compared to negative control (A549 ​cells). (F) Fold changes in gene (Interferon regulatory factor 1; IRF1 and IRF7) expression based on qRT-PCR in NSP1 and NSP15 stably expressing A549 ​cells lines over negative control (A549 ​cells). Non-parametric Wilcoxon tests (Mann-Whitney) was used to assess normal distribution and test significance with the results shown as mean ​± ​SD. ∗ ( p ​≤ ​0.05), and ∗∗∗ ( p ​≤ ​0.005) indicates a statistically significant difference. All experiments were performed in 3 technical replicates, and the data are representative of results from minimum of 3 independent experiments.

Article Snippet: Cells were washed with phosphate buffered saline (PBS) buffer and pre-treated for 2 ​h with the following TLR ligands: TLR2 ligand PAM3CSK4 (0.005, 0.05, 0.5 and 5 ​μg/mL; Invivogen, CA), TLR3 ligand Polyinosinic:polycytidylic acid (polyI:C) (0.005, 0.05, 0.5 and 5 ​μg/mL; Invivogen, CA), TLR4 ligand lipopolysaccharides from Escherichia coli O111:B4 (LPS) (0.01, 0.1, 1.0, 10 and 50 ​μg/mL; Millipore-Sigma, CA) and TLR9 ligand CpG ODN 2007 (0.005, 0.05, 0.5, 5 and 50 ​μg/mL, Life Technologies, CA). (ii) Infection and Readout: A549 ​cells and stably expressing cell lines were infected (multiplicity of infection; MOI of 0.4) with the vesicular stomatitis virus (VSV)-EGFP and incubated (37 ​°C and 5% CO 2 ) for 24 ​h.

Techniques: Expressing, Stable Transfection, Negative Control, Quantitative RT-PCR, MANN-WHITNEY

Journal: Current Research in Virological Science

Article Title: The severe acute respiratory syndrome coronavirus 2 non-structural proteins 1 and 15 proteins mediate antiviral immune evasion

doi: 10.1016/j.crviro.2022.100021

Figure Lengend Snippet: TLR ligand sensitivity of NSP1 and NSP15 stably expressing A549 cells. Replication efficiency of the interferon sensitive VSV-EGFP virus in A549 ​cells. Analysis of VSV-EGFP (moi ​= ​0.4) virus replication in NSP1 and NSP15 stably expressing A549 ​cells lines compared to control transfected (pCDNA3.1/V5–HIS-TOPO) or negative (A549 ​cells) based on fluorescence (EGFP; excitation 490 ​nm). Cell lines were pre-stimulated (2 ​h) with (A) TLR3 ligand POLY; IC (0.005, 0.05, 0.5 and 5 ​μg/mL), (B) TLR9 ligand CpG ODN 2007 (0.005, 0.05, 0.5 and 5 ​μg/mL), (C) TLR2 ligand PAM3CSK4 (0.005, 0.05, 0.5 and 5 ​μg/mL), and (D) TLR4 ligand LPS (0.01, 0.1, 1.0, 10 and 50 ​μg/mL) prior to infection with VSV-EGFP. Fold changes in (E) IRF1 and IRF7, (F) PAN-IFN-α, IFN-β and IFN-γ, and (G) ISG-15, ISG-54, ISG-56, Viperin, OAS, PKR, IFIT1 and IFITM-3 gene expression based on qRT-PCR in NSP1 and NSP15 stably expressing A549 ​cells lines over negative control (A549 ​cells) stimulated for 2 ​h with 5 ​μg/mL CpG ODN 2007. The mean fluorescence unit in uninfected cells was used as the normalization factor for VSV-EGFP readout in infected A549 ​cells.Non-parametric Wilcoxon tests (Mann-Whitney) and one-way ANOVA was used to assess normal distribution and test significance with the results shown as mean ​± ​SD. ∗∗ ( p ​≤ ​0.01), ∗∗∗ ( p ​≤ ​0.005) and ∗∗∗∗ ( p ​≤ ​0.001) indicates a statistically significant difference. All viral infection experiments were performed in 10 technical replicates, and the data are representative of results from 3 independent experiments.

Article Snippet: Cells were washed with phosphate buffered saline (PBS) buffer and pre-treated for 2 ​h with the following TLR ligands: TLR2 ligand PAM3CSK4 (0.005, 0.05, 0.5 and 5 ​μg/mL; Invivogen, CA), TLR3 ligand Polyinosinic:polycytidylic acid (polyI:C) (0.005, 0.05, 0.5 and 5 ​μg/mL; Invivogen, CA), TLR4 ligand lipopolysaccharides from Escherichia coli O111:B4 (LPS) (0.01, 0.1, 1.0, 10 and 50 ​μg/mL; Millipore-Sigma, CA) and TLR9 ligand CpG ODN 2007 (0.005, 0.05, 0.5, 5 and 50 ​μg/mL, Life Technologies, CA). (ii) Infection and Readout: A549 ​cells and stably expressing cell lines were infected (multiplicity of infection; MOI of 0.4) with the vesicular stomatitis virus (VSV)-EGFP and incubated (37 ​°C and 5% CO 2 ) for 24 ​h.

Techniques: Stable Transfection, Expressing, Transfection, Fluorescence, Infection, Quantitative RT-PCR, Negative Control, MANN-WHITNEY

Journal: Current Research in Virological Science

Article Title: The severe acute respiratory syndrome coronavirus 2 non-structural proteins 1 and 15 proteins mediate antiviral immune evasion

doi: 10.1016/j.crviro.2022.100021

Figure Lengend Snippet: Identification of the NSP1 protein functional domain. Truncation of NSP1 limits VSV-EGFP virus replication. (A) Schematic diagram depicting the full length of NSP1 and truncated NSP1 1/3 and NSP1 2/3 in amino acid. The SARS-CoV-2 NSP1 was truncated by high fidelity PCR (GoTaq) and subcloned into a pCDNA3.1/V5–HIS-TOPO plasmid ( Hin dIII and Eco RV insertion sites) which was transfected into A549 ​cells to generate stably expressing cell lines. Expression were confirmed by total RNA extraction (polymerase chain reaction; PCR) and total protein extraction (Western blot). (B) Representative pictures of confocal microscopy imaging with maximum projections of z-stacks for each channel demonstrating the subcellular localization of expressed full length of NSP1, NSP1 1/3 and NSP1 2/3 (anti-V5 antibody; 488 ​nm) in stably expressing A549 ​cells. Analysis of VSV-EGFP (moi ​= ​0.4) virus replication in (C) NSP1, NSP1 1/3 and NSP1 2/3 stably expressing A549 ​cells lines compared to control transfected (pCDNA3.1/V5–HIS-TOPO) or negative (A549 ​cells) based on fluorescence (EGFP; excitation 490 ​nm). Representative overlapping FACS histogram demonstrating the expression of (D) TLR2 (E) TLR3, (F) TLR4 and (G) TLR9 as gated on live (7AAD-) NSP1, NSP1 1/3 and NSP1 2/3 stably expressing A549 ​cells to analyze (H) the number of cells expressing TLR2, TLR3, TLR4 and TLR9 compared to negative control (A549 ​cells). Analysis of VSV-EGFP (moi ​= ​0.4) virus replication in NSP1, NSP1 1/3 and NSP1 2/3 stably expressing A549 ​cells lines compared to control transfected (pCDNA3.1/V5–HIS-TOPO) or negative (A549 ​cells) based on fluorescence (EGFP; excitation 490 ​nm) pre-stimulated (2 ​h) with (I) TLR3 ligand POLY; IC (0.005, 0.05, 0.5 and 5 ​μg/mL), (J) TLR9 ligand CpG ODN 2007 (0.005, 0.05, 0.5 and 5 ​μg/mL), (K) TLR4 ligand LPS (0.01, 0.1, 1.0, 10 and 50 ​μg/mL) and (L) TLR2 ligand PAM3CSK4 (0.005, 0.05, 0.5 and 5 ​μg/mL) and prior to infection with VSV-EGFP. The mean fluorescence unit in uninfected cells was used as the normalization factor for VSV-EGFP readout in infected A549 ​cells.Non-parametric Wilcoxon tests (Mann-Whitney) and one-way ANOVA was used to assess normal distribution and test significance with the results shown as mean ​± ​SD. ∗ ( p ​≤ ​0.05), ​∗∗ ( p ​≤ ​0.01), ∗∗∗ ( p ​≤ ​0.005) and ∗∗∗∗ ( p ​≤ ​0.001) indicates a statistically significant difference. All viral infection experiments were performed in 10 technical replicates, and the data are representative of results from 3 independent experiments.

Article Snippet: Cells were washed with phosphate buffered saline (PBS) buffer and pre-treated for 2 ​h with the following TLR ligands: TLR2 ligand PAM3CSK4 (0.005, 0.05, 0.5 and 5 ​μg/mL; Invivogen, CA), TLR3 ligand Polyinosinic:polycytidylic acid (polyI:C) (0.005, 0.05, 0.5 and 5 ​μg/mL; Invivogen, CA), TLR4 ligand lipopolysaccharides from Escherichia coli O111:B4 (LPS) (0.01, 0.1, 1.0, 10 and 50 ​μg/mL; Millipore-Sigma, CA) and TLR9 ligand CpG ODN 2007 (0.005, 0.05, 0.5, 5 and 50 ​μg/mL, Life Technologies, CA). (ii) Infection and Readout: A549 ​cells and stably expressing cell lines were infected (multiplicity of infection; MOI of 0.4) with the vesicular stomatitis virus (VSV)-EGFP and incubated (37 ​°C and 5% CO 2 ) for 24 ​h.

Techniques: Functional Assay, Plasmid Preparation, Transfection, Stable Transfection, Expressing, RNA Extraction, Polymerase Chain Reaction, Protein Extraction, Western Blot, Confocal Microscopy, Imaging, Fluorescence, Negative Control, Infection, MANN-WHITNEY

Journal: Current Research in Virological Science

Article Title: The severe acute respiratory syndrome coronavirus 2 non-structural proteins 1 and 15 proteins mediate antiviral immune evasion

doi: 10.1016/j.crviro.2022.100021

Figure Lengend Snippet: Identification of the NSP15 protein functional domain. Truncation of NSP15 limits VSV-EGFP virus replication. (A) Schematic diagram depicting the full length of NSP15 and truncated NSP15 1/3 and NSP15 2/3 in amino acid. The SARS-CoV-2 NSP15 was truncated by high fidelity PCR (GoTaq) and subcloned into a pCDNA3.1/V5–HIS-TOPO plasmid ( Hin dIII and Eco RV insertion sites) which was transfected into A549 ​cells to generate stably expressing cell lines. Expression were confirmed by total RNA extraction (polymerase chain reaction; PCR) and total protein extraction (Western blot). (B) Representative pictures of confocal microscopy imaging with maximum projections of z-stacks for each channel demonstrating the subcellular localization of expressed full length of NSP15, NSP15 1/3 and NSP15 2/3 (anti-V5 antibody; 488 ​nm) in stably expressing A549 ​cells. Analysis of VSV-EGFP (moi ​= ​0.4) virus replication in (C) NSP15, NSP15 1/3 and NSP15 2/3 stably expressing A549 ​cells lines compared to control transfected (pCDNA3.1/V5–HIS-TOPO) or negative (A549 ​cells) based on fluorescence (EGFP; excitation 490 ​nm). Representative overlapping FACS histogram demonstrating the expression of (D) TLR2 (E) TLR3, (F) TLR4 and (G) TLR9 as gated on live (7AAD-) NSP15, NSP15 1/3 and NSP15 2/3 stably expressing A549 ​cells lines cells to analyze (H) the number of cells expressing TLR2, TLR3, TLR4 and TLR9 compared to negative control (A549 ​cells). Analysis of VSV-EGFP (moi ​= ​0.4) virus replication in NSP15, NSP15 1/3 and NSP15 2/3 stably expressing A549 ​cells lines compared to control transfected (pCDNA3.1/V5–HIS-TOPO) or negative (A549 ​cells) based on fluorescence (EGFP; excitation 490 ​nm) pre-stimulated (2 ​h) with (I) TLR3 ligand polyI:C (0.005, 0.05, 0.5 and 5 ​μg/mL), (J) TLR9 ligand CpG ODN 2007 (0.005, 0.05, 0.5 and 5 ​μg/mL), (K) TLR4 ligand LPS (0.01, 0.1, 1.0, 10 and 50 ​μg/mL) and (L) TLR2 ligand PAM3CSK4 (0.005, 0.05, 0.5 and 5 ​μg/mL) and prior to infection with VSV-EGFP. The mean fluorescence unit in uninfected cells was used as the normalization factor for VSV-EGFP readout in infected A549 ​cells. Non-parametric Wilcoxon tests (Mann-Whitney) or one-way ANOVA was used to assess normal distribution and test significance with the results shown as mean ​± ​SD. ∗ ( p ​≤ ​0.05), ​∗∗ ( p ​≤ ​0.01), ∗∗∗ ( p ​≤ ​0.005) and ∗∗∗∗ ( p ​≤ ​0.001) indicates a statistically significant difference. All viral infection experiments were performed in 10 technical replicates, and the data are representative of results from 3 independent experiments.

Article Snippet: Cells were washed with phosphate buffered saline (PBS) buffer and pre-treated for 2 ​h with the following TLR ligands: TLR2 ligand PAM3CSK4 (0.005, 0.05, 0.5 and 5 ​μg/mL; Invivogen, CA), TLR3 ligand Polyinosinic:polycytidylic acid (polyI:C) (0.005, 0.05, 0.5 and 5 ​μg/mL; Invivogen, CA), TLR4 ligand lipopolysaccharides from Escherichia coli O111:B4 (LPS) (0.01, 0.1, 1.0, 10 and 50 ​μg/mL; Millipore-Sigma, CA) and TLR9 ligand CpG ODN 2007 (0.005, 0.05, 0.5, 5 and 50 ​μg/mL, Life Technologies, CA). (ii) Infection and Readout: A549 ​cells and stably expressing cell lines were infected (multiplicity of infection; MOI of 0.4) with the vesicular stomatitis virus (VSV)-EGFP and incubated (37 ​°C and 5% CO 2 ) for 24 ​h.

Techniques: Functional Assay, Plasmid Preparation, Transfection, Stable Transfection, Expressing, RNA Extraction, Polymerase Chain Reaction, Protein Extraction, Western Blot, Confocal Microscopy, Imaging, Fluorescence, Negative Control, Infection, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: CAL-1 as Cellular Model System to Study CCR7-Guided Human Dendritic Cell Migration

doi: 10.3389/fimmu.2021.702453

Figure Lengend Snippet: Phenotypic characterization of human CAL-1 cells and CCR7 induction upon exposure to GM-CSF and maturation by R848. (A) Surface expression of CD123, CD11c, CD40, CD86, HLA-DR and CCR7 (solid lines) on CAL-1 cells that were stimulated or not for 18-19h with the TLR9 ligand CpG-B ODN 2006 (1μM) or the TLR7/8 ligand Resiquimod R848 (10μg/ml). Representative flow cytometry histograms derived from one out of three independent experiments are depicted. Unstained cells or isotype-matched controls for CCR7 stainings are shown as dashed lines. (B) Surface expression of CD123, CD11c, CD40, CD86, HLA-DR and CCR7 on CAL-1 cells cultured for 3 days in the presence of 10ng/ml GM-CSF. Where indicated, these GM/CAL-1 cells were in addition matured by either CpG-B or R848 as in (A) . Representative flow cytometry histograms derived from one out of three independent experiments are depicted. (C) Quantitative analysis of surface markers on CAL-1 (white bars) and GM/CAL-1 (blue bars) cells. Mean values ± SEM of the 3 independent experiments (A, B) are shown.

Article Snippet: Where indicated, CAL-1 cells (2x 10 5 cells/ml) were exposed to 10ng/ml human GM-CSF (Peprotech, LuBioScience; catalog #300-03) for 3 days, referred to as GM/CAL-1 cells, washed, resuspended at a density of 1x 10 6 cells/ml in complete growth medium without GM-CSF and then left untreated or matured for another 18-19h with either the TLR7/8 agonist Resiquimod R848 (10μg/ml; Sigma–Aldrich, Buchs, Switzerland; #SML0196) or the TLR9 ligand CpG-B ODN 2006 (1μM; InvivoGen; LabForce, Muttenz, Switzerland; #tlrl-2006-1).

Techniques: Expressing, Flow Cytometry, Derivative Assay, Cell Culture

Journal: Clinical & Translational Immunology

Article Title: Next‐generation Bruton's tyrosine kinase inhibitor BIIB091 selectively and potently inhibits B cell and Fc receptor signaling and downstream functions in B cells and myeloid cells

doi: 10.1002/cti2.1295

Figure Lengend Snippet: BIIB091 blocks B‐cell proliferation and antigen‐presenting functions in vitro and the associated B‐cell activation transcriptional programme. (a, b) Pseudocolor plots displaying side scatter against the dilution of CFSE used as a measure of proliferation of human B cells left either unstimulated or stimulated for 5 days with anti‐IgM alone, anti‐IgM with CD40L and IL‐4, anti‐IgM with CD40L and IL‐21, or CpG ODN 2006 in the presence or absence of 100 n m BIIB091 (a) . Percent inhibition of B‐cell proliferation (relative to proliferation level observed in DMSO control condition) obtained from six independent experiments (individual data points) and measured either three (filled circle), four (square) or five (open circle) days post‐stimulation (b) . (c) Log 2 fold‐change × fold‐change plot of differentially expressed genes (FDR < 0.05; fold change > |1.2|) observed when comparing anti‐IgM, CD40L and IL‐21‐stimulated human B cells to unstimulated B cells on the x ‐axis and anti‐IgM, CD40L and IL‐21‐stimulated B cells treated with 100 n m BIIB091 to stimulated B cells treated with DMSO on the y ‐axis. Data from five independent experiments. (d) Log 2 fold‐change × fold‐change plot of differentially expressed genes (FDR < 0.05; fold change > |1.2|) observed when comparing anti‐IgM, CD40L and IL‐4‐stimulated human B cells to unstimulated B cells on the x ‐axis and anti‐IgM, CD40L and IL‐4 stimulated B cells treated with 100 n m BIIB091 to stimulated B cells treated with DMSO on the y ‐axis. Data from five independent experiments. (e–g) Pseudocolor plots displaying side scatter against the dilution of CellTrace Violet used as a measure of proliferation of mouse OTII CD4 + T cell (expressing transgenic T‐cell receptor specific for the ovalbumin (OVA) peptide 323–339) upon B‐cell receptor targeting of OVA protein antigen to B cells using antibodies directed against the B‐cell receptor (e) or upon addition of OVA peptide 323–339 (g) to co‐cultures of splenic B cells and OTII T cells in the presence or absence of titrating concentrations of BIIB091 (left). Dot plot shows the per cent of proliferating OVA 323–339 ‐specific CD4 + T versus the concentration of BIIB091 (right). IC 50 values obtained with B‐cell receptor targeting of OVA protein antigen to B cells in three independent experiments are shown along with the mean and SD (22 ± 8 n m ) (f) .

Article Snippet: Labelled B cells were then stimulated with TLR9 ligand CpG ODN 2006 (Invivogen, San Diego, CA) or F(ab′)2 goat anti‐human IgM (1 µg mL −1 ; Jackson ImmunoResearch) or combinations of anti‐IgM)/MEGACD40L (1 μg mL −1 ; Enzo Life Sciences, Farmingdale, NY) with either recombinant human IL‐4 (20 ng mL −1 ; R&D Systems) or recombinant human IL‐21 (20 ng mL −1 ; Miltenyi Biotec) in the presence or absence of 100 n m of BIIB091 in serum‐free X‐VIVO 10™ medium for 3–5 days.

Techniques: In Vitro, Activation Assay, Inhibition, Expressing, Transgenic Assay, Concentration Assay