Journal: Nature immunology

Article Title: Genetic dissection of TLR9 reveals complex regulatory and cryptic proinflammatory roles in mouse lupus.

doi: 10.1038/s41590-022-01310-2

Figure Lengend Snippet: Fig. 4 | Effects of TLR9 and sex on TLR7 expression and distribution. a–d, Intracellular staining of TLR7 and TLR9 (ratio of TLR7 MFI over average TLR7 MFI of the corresponding male or female WT group) in FO (a,c) and MZ (b,d) B cells of Tlr9-mutant male or female BALB/c mice. Data points indicate individual mice (Tlr9K51E/K51E male, n = 7; female, n = 11; Tlr9 WT male, n = 7, female, n = 11; Tlr9P915H/P915H male, n = 7, female, n = 10; Tlr9+/− male, n = 3, female, n = 7; Tlr9−/− male, n = 7, female, n = 11), and bars indicate the mean ± s.e.m. of two or three experiments pooled (except from one experiment for Tlr9+/− males); *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test; female, Tlr9K51E/K51E versus Tlr9−/−, P = 0.0026; Tlr9−/− versus Tlr9P915H/P915H, P = 0.0251; Tlr9WT versus Tlr9−/−, P = 0.0003 (a); female, Tlr9K51E/K51E versus Tlr9−/−, P = 0.0170; Tlr9WT versus Tlr9P915H/

Article Snippet: Cells were stained at 50–100 million cells per ml overnight at 4 °C in 1× Perm/Wash buffer with fluorochrome-conjugated antibodies CD45R (clone RA3-6B2, 1:400), TLR7 (clone A94B10, 1:800), TLR9–biotin (clone Nar9, from K. Miyake67, biotin conjugation in-house, 1:600) and unconjugated EEA1 (polyclonal anti-rabbit, Novus, 1:100) or LAMP-1 (polyclonal anti-rabbit, Abcam, 1:100).

Techniques: Expressing, Staining, Mutagenesis

Journal: PLoS ONE

Article Title: Cholecalciferol decreases inflammation and improves vitamin D regulatory enzymes in lymphocytes in the uremic environment: A randomized controlled pilot trial

doi: 10.1371/journal.pone.0179540

Figure Lengend Snippet: Expression of TLR7, TLR9, IL-6, IFN-γ, VDR, CYP27b1 and CYP24a1 in B and T lymphocytes.

Article Snippet: After lysing, cells were permeabilized as described above to intracellular stain with 5 μL pf FITC-labeled IL-6 (BD Biosciences, San Diego, USA), 10 μL of PE-labeled TLR7 antibody (R&D Systems, Mineapolis, USA), 5 μL of APC-Cy7-labeled IFN-γ (eBiosciences, San Diego, USA) and 10 μL of APC-labeled TLR9 (BD Biosciences, San Diego, USA) antibodies.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Cholecalciferol decreases inflammation and improves vitamin D regulatory enzymes in lymphocytes in the uremic environment: A randomized controlled pilot trial

doi: 10.1371/journal.pone.0179540

Figure Lengend Snippet: Effect of 25 and 1,25 vitamin D on TLR7 and TLR9 expression (MFI) in B lymphocytes (Figs A and B) and T lymphocytes (Figs C and D) in presence of healthy or uremic serum (US) and after CYP24 silencing (siRNA).

Article Snippet: After lysing, cells were permeabilized as described above to intracellular stain with 5 μL pf FITC-labeled IL-6 (BD Biosciences, San Diego, USA), 10 μL of PE-labeled TLR7 antibody (R&D Systems, Mineapolis, USA), 5 μL of APC-Cy7-labeled IFN-γ (eBiosciences, San Diego, USA) and 10 μL of APC-labeled TLR9 (BD Biosciences, San Diego, USA) antibodies.

Techniques: Expressing

Journal: Journal of Innate Immunity

Article Title: TLR7 Activation in M-CSF-Dependent Monocyte-Derived Human Macrophages Potentiates Inflammatory Responses and Prompts Neutrophil Recruitment

doi: 10.1159/000530249

Figure Lengend Snippet: Regulated expression of TLR7 in human macrophages. a Schematic representation of the in vitro generation of M-MØ and GM-MØ from peripheral blood monocytes. TLR7 gene expression (normalized fluorescence intensity) in M-MØ and GM-MØ, as determined in microarray experiments (GSE68061) on three independent samples. Statistically significant differences (adjp) are indicated. b TLR7 protein levels in four independent preparations of M-MØ and GM-MØ, as determined by Western blot (left panel). Relative level of TLR7, including both the full-length (140 kDa) and the proteolytically processed mature (75 kDa) forms, were determined in M-MØ and GM-MØ by densitometry. Mean ± SEM of four independent samples is shown (*** p < 0.001). c Relative TLR7 gene expression in Monocytes, M-MØ and GM-MØ, as determined by RNA-sequencing (GSE188278) on three independent samples. Statistically significant differences are indicated. d TLR7 gene expression in M-MØ before and after exposure to GM-CSF (1,000 U/mL) for 24 or 48 h, and either with or without medium replacement, as determined by RT-PCR. Mean and SEM of four independent experiments are shown (** p < 0.005). e TLR7 gene expression in M-MØ generated from control individuals (2 independent donors) or an MCTO patient (two distinct samples derived from a single MCTO patient), as determined by RNA-sequencing (GSE155883). Statistically significant differences are indicated. f TLR7 gene expression in M-MØ and GM-MØ before and after LPS activation, as determined in microarray experiments (GSE99056) on three independent samples. Statistically significant differences (adjp) are indicated. g t-Distributed stochastic neighbor embedding plots (t-SNE, Perplexity:25, Color plot by k = 10) illustrating TLR7 , MAFB , and FOLR2 gene expression in the human fetal-maternal interface ( https://www.ebi.ac.uk/gxa/sc/home ). The “Expression level” bar indicates the expression level in each case (Smartseq 2 data; CPM, counts per million).

Article Snippet: Western blot was carried out following previously described procedures and using antibodies against TLR7 (#5632, Cell Signaling), MAFB (HPA005653, Sigma-Aldrich), MAF (sc-7866; Santa Cruz Biotechnology), AhR (sc-133088, Santa Cruz Biotechnology), phosphorylated and total ERK1/2, p38MAPK, and JNK (Cell Signaling), phosphorylated STAT1 and STAT3 (BD Biosciences), IκBα, and phosphorylated CREB (Cell Signaling).

Techniques: Expressing, In Vitro, Gene Expression, Fluorescence, Microarray, Western Blot, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Generated, Control, Derivative Assay, Activation Assay

Journal: Journal of Innate Immunity

Article Title: TLR7 Activation in M-CSF-Dependent Monocyte-Derived Human Macrophages Potentiates Inflammatory Responses and Prompts Neutrophil Recruitment

doi: 10.1159/000530249

Figure Lengend Snippet: TLR7 conditions the M-MØ responses to later stimuli. a Schematic representation of the treatment of M-MØ with CL264 (1 μg/mL) for analysis of changes in the gene expression profile. b Relative expression of the indicated genes of the M-MØ-specific “anti-inflammatory gene set” (blue) and GM-MØ-specific “pro-inflammatory gene set” (red) in untreated (−) and CL264-treated M-MØ, as determined by quantitative RT-PCR on six macrophage samples derived from six independent donors. Mean ± SEM are shown (* p < 0.05). c GSEA of the GM-MØ-specific “pro-inflammatory gene set” (left panel) or M-MØ-specific “anti-inflammatory gene set” (right panel) on the ranked comparison of CL264-treated M-MØ and untreated M-MØ at 12 h after stimulation (GSE156921). Normalized Enrichment Score (NES) and FDRq values are indicated. d Schematic representation of the sequential treatment of CL264-treated with CL264 (1 μg/mL) or LPS (10 ng/mL) for analysis of intracellular signaling (30 or 60 min) and cytokine production (after 16 h). e Levels of phosphorylated ERK (p-ERK), phosphorylated JNK (p-JNK), phosphorylated STAT3 (p-STAT3), phosphorylated STAT1 (p-STAT1), and IκBα in M-MØ exposed to the indicated primary and secondary stimuli after 30 min (p-ERK, p-JNK, IkBα) or 60 min (p-STAT1, p-STAT3) of stimulation, as determined by Western blot. In each case, four macrophage samples derived from four independent donors were analyzed, and a representative experiment is shown. Protein loading was normalized after determination of the level of vinculin in each case. f Production of the indicated cytokines in M-MØ was evaluated after exposure to the indicated primary (24 h) and secondary (16 h) stimuli, as determined by ELISA. Mean ± SEM of four independent donors are shown (* p < 0.05; ** p < 0.01).

Article Snippet: Western blot was carried out following previously described procedures and using antibodies against TLR7 (#5632, Cell Signaling), MAFB (HPA005653, Sigma-Aldrich), MAF (sc-7866; Santa Cruz Biotechnology), AhR (sc-133088, Santa Cruz Biotechnology), phosphorylated and total ERK1/2, p38MAPK, and JNK (Cell Signaling), phosphorylated STAT1 and STAT3 (BD Biosciences), IκBα, and phosphorylated CREB (Cell Signaling).

Techniques: Gene Expression, Expressing, Quantitative RT-PCR, Derivative Assay, Comparison, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of Innate Immunity

Article Title: TLR7 Activation in M-CSF-Dependent Monocyte-Derived Human Macrophages Potentiates Inflammatory Responses and Prompts Neutrophil Recruitment

doi: 10.1159/000530249

Figure Lengend Snippet: TLR7 activation of M-MØ induces neutrophil-attracting chemokines. a k-means clustering of the 467 genes differentially expressed (|log2FC|>3) at any time point between LPS-treated M-MØ and CL264-treated M-MØ (GSE156921). For each gene, expression levels in the three donors are represented after normalizing gene expression and k-means clustering using Genesis ( http://genome.tugraz.at/genesisclient/ ). The identity of some genes within each cluster is indicated. b Heatmap of the expression of the indicated chemokine-encoding genes in LPS-treated M-MØ and CL264-treated M-MØ at the indicated time points after stimulation (GSE156921). c GSEA of the “GOBP_NEUTROPHIL_CHEMOTAXIS” gene set on the ranked comparison of the transcriptomes of CL264-treated M-MØ and untreated M-MØ at 12 h after stimulation (GSE156921). Normalized Enrichment Score (NES) and FDRq values are indicated. d Production of the indicated chemokines in untreated (−) M-MØ, LPS-treated (10 ng/mL) and CL264-treated (1 μg/mL) M-MØ (16 h), as determined by ELISA. Mean ± SEM of four independent donors are shown (* p < 0.05). e Neutrophil migration in response to IL-8 or culture supernatants from untreated M-MØ or CL264-treated (1 μg/mL) M-MØ, as determined by Transwell migration assays. Mean ± SEM of four independent donors are shown (** p < 0.01). f Production of the indicated cytokines in M-MØ was evaluated after exposure to the indicated primary stimulus (24 h) and secondary stimulus (16 h), as determined by ELISA. Mean ± SEM of four independent donors are shown (** p < 0.01).

Article Snippet: Western blot was carried out following previously described procedures and using antibodies against TLR7 (#5632, Cell Signaling), MAFB (HPA005653, Sigma-Aldrich), MAF (sc-7866; Santa Cruz Biotechnology), AhR (sc-133088, Santa Cruz Biotechnology), phosphorylated and total ERK1/2, p38MAPK, and JNK (Cell Signaling), phosphorylated STAT1 and STAT3 (BD Biosciences), IκBα, and phosphorylated CREB (Cell Signaling).

Techniques: Activation Assay, Gene Expression, Expressing, Chemotaxis Assay, Comparison, Enzyme-linked Immunosorbent Assay, Migration

Journal: Journal of Innate Immunity

Article Title: TLR7 Activation in M-CSF-Dependent Monocyte-Derived Human Macrophages Potentiates Inflammatory Responses and Prompts Neutrophil Recruitment

doi: 10.1159/000530249

Figure Lengend Snippet: The acquisition of the TLR7-specific transcriptome and neutrophil-recruiting chemokine production depends on the macrophage polarization state. a Schematic representation of siRNA-mediated gene silencing before treatment of M-MØ with CL264 (1 μg/mL) for determination of the gene expression profile. b Summary of GSEA of the Clusters identified in on the ranked comparison of the transcriptomes of CL264-treated (4 h) siAHR M-MØ (siAHR M-MØ + CL264 [ left panel ]), siMAF M-MØ (siMAF M-MØ + CL264 [ middle panel ]), or siMAFB M-MØ (siMAFB M-MØ + CL264 [ right panel ]) versus siCNT M-MØ + CL264. FDRq value is indicated in each case. c Relative expression of the indicated chemokine-encoding genes in CL264-treated (4 h) siAHR M-MØ, siMAF M-MØ, and siMAFB M-MØ (log2FC [siTF-M-MØ + CL264/siCNT-M-MØ + CL264]), as determined by RNA-Seq (*, adjp < 0.05). d Schematic representation of siRNA-mediated gene silencing before treatment of M-MØ with CL264 (1 μg/mL) for determination of the cytokine profile. e Production of the indicated chemokines in untreated M-MØ (−), siCNT M-MØ + CL264, siAHR M-MØ + CL264, siMAF M-MØ + CL264, and siMAFB M-MØ + CL264, as determined by ELISA. Data represent the production of chemokines by each cell type relative to the levels produced by siCNT M-MØ + CL264. Mean ± SEM of three independent donors are shown (* p < 0.05; ** p < 0.01).

Article Snippet: Western blot was carried out following previously described procedures and using antibodies against TLR7 (#5632, Cell Signaling), MAFB (HPA005653, Sigma-Aldrich), MAF (sc-7866; Santa Cruz Biotechnology), AhR (sc-133088, Santa Cruz Biotechnology), phosphorylated and total ERK1/2, p38MAPK, and JNK (Cell Signaling), phosphorylated STAT1 and STAT3 (BD Biosciences), IκBα, and phosphorylated CREB (Cell Signaling).

Techniques: Gene Expression, Comparison, Expressing, RNA Sequencing, Enzyme-linked Immunosorbent Assay, Produced

Journal: BMC Complementary Medicine and Therapies

Article Title: Huang Lian Jie Du Decoction enhances the anti-tumor efficacy of immune checkpoint inhibitors by activating TLR7/8 signalling in melanoma

doi: 10.1186/s12906-024-04444-y

Figure Lengend Snippet: Toll-like receptor signaling pathway is activated after HLJD treatment. ( A ) RNA-seq analysis was performed and the volcano plot was demonstrated. ( n = 3 mice/group) ( B ) Interaction net of the significant pathways (Path-Net) of differentially signaling pathways. ( C ) Immunohistochemistry was used to analyze the expression levels of TLR7 (400×). HLJD = Huang Lian Jie Du Decoction, ICIs = anti-PD-1 + anti-CTLA-4

Article Snippet: Primary antibody against mouse TLR7 was purchased from Proteintech (Wuhan, China), and secondary antibodies and DAPI were obtained from Servicebio (Wuhan, China).

Techniques: RNA Sequencing, Protein-Protein interactions, Immunohistochemistry, Expressing

Journal: BMC Complementary Medicine and Therapies

Article Title: Huang Lian Jie Du Decoction enhances the anti-tumor efficacy of immune checkpoint inhibitors by activating TLR7/8 signalling in melanoma

doi: 10.1186/s12906-024-04444-y

Figure Lengend Snippet: HLJD treatment upregulated Type I IFN signaling. ( A , B and C ) Volcano Plot graph and Bar graphs of PCR-array of TLR7/8 and Type I IFN signaling axis. ( D ) The expression of IRF7 was determined by qRT-PCR. ( E ) IFN-α quantification by ELISA in tumor tissue. Statistical differences were determined using the student’s t-test. (* p < 0.05, ** p < 0.01) HLJD = Huang Lian Jie Du Decoction, ICIs = anti-PD-1 + anti-CTLA-4

Article Snippet: Primary antibody against mouse TLR7 was purchased from Proteintech (Wuhan, China), and secondary antibodies and DAPI were obtained from Servicebio (Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay