Journal: iScience

Article Title: Evidence against the role of toll-like receptors 7 and 8 in sex selection in mice, cattle, and humans

doi: 10.1016/j.isci.2025.113164

Figure Lengend Snippet: Effects of TLR7/8 ligand (R848) on murine sperm partitioning and In vitro fertilization (IVF) (A) The schematic diagram of R848 treatment and swim-up layer assignment. (B) The percentages of sperm in different layers after treatment with R848 at various concentrations, ns = not significant ( p > 0.05). (C) The changes in the percentages of sperm of the same individual mice before and after R848 treatment in each swim-up layer. Data in (B and C) were analyzed using one-way ANOVA. (D and E) Cleavage (D) and blastocyst (E) rates of embryos derived from R848-treated sperm of different layers. Data were analyzed with one-way ANOVA followed by Tukey’s post-hoc tests. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01. (F) The percentages of male embryos derived from IVF using sperm treated with 0 or 0.03 μM R848. The expected male embryo ratio was 50% (red bar). All values are mean ± SD of at least three replicates. (G) Representative agarose gel images of embryo sex determination by PCR. The product sizes for SRY and XIST were 105 and 147 bp, respectively. The bands below the specific ones were likely primer-dimers. Each lane represented an individual blastocyst. Lane marked with “F” and “M” were deemed to be female and male embryos, respectively.

Article Snippet: TLR7 polyclonal antibody , Bioss , Cat# bs-6601R; RRID:AB_11090775.

Techniques: In Vitro, Derivative Assay, Agarose Gel Electrophoresis

Journal: iScience

Article Title: Evidence against the role of toll-like receptors 7 and 8 in sex selection in mice, cattle, and humans

doi: 10.1016/j.isci.2025.113164

Figure Lengend Snippet: TLR7/8 ligand (R848) treatment did not separate X- and Y- sperm (A) Validation of TaqMan real-time PCR for sex chromosome ratio determination by using different ratios of male vs. female mouse genomic DNA. The expected ratios are marked with red bars. Data were analyzed with one sample t test. (B) TaqMan real-time PCR determination of the X-sperm ratios in different layers of sperm treated with R848. Data were analyzed with one-way ANOVA, ns = not significant ( p > 0.05). (C) Schematic diagram of centrifugation of sperm before R848 treatment and swim-up. (D) TaqMan real-time PCR determination of the X-sperm ratios in different layers of sperm from (C). Data were analyzed with one-way ANOVA. All values are mean ± SD from at least three replicates.

Article Snippet: TLR7 polyclonal antibody , Bioss , Cat# bs-6601R; RRID:AB_11090775.

Techniques: Biomarker Discovery, Real-time Polymerase Chain Reaction, Centrifugation

Journal: iScience

Article Title: Evidence against the role of toll-like receptors 7 and 8 in sex selection in mice, cattle, and humans

doi: 10.1016/j.isci.2025.113164

Figure Lengend Snippet: Localization of TLR7/8 in murine sperm (A) Immunofluorescence staining of TLR7/8 (green), acetyl α-tubulin (green), and DNA (blue) in murine caudal epididymal sperm. (i) The Bioss antibody against TLR7 stained the whole sperm tails; (ii) The Abcam antibody against TLR7 stained the lower half of the sperm tail as well as the acrosome of the sperm (red arrow); (iii) TLR8 antibody stained either the lower half (yellow arrow) or the entire sperm tail (blue arrow), as well as the acrosome (red arrow). Scale bar, 25 μm. (B) The percentages of murine sperm stained positively for TLR7/8 by immunofluorescence. (C) Flow cytometry analysis of mouse sperm stained with TLR7/8 and DAPI. In controls (left panels) the sperm were probed with the secondary antibody only and provided the cutoff (vertical lines in the upper panels) for stained and unstained sperm. The cells were detected by the FITC-A ( x axis) and DAPI ( y axis) channels. Each dot represents one sperm. The upper panel showed the distribution of the positively and negatively stained sperm, the lower panel displayed the intensity of fluorescence of the sperm ( x axis) and sperm counts ( y axis). The middle and right panels represent sperm stained for TLR7 and TLR8, respectively. Nearly all sperm were stained by both antibodies. (D) The percentages of mouse sperm positively stained for TLR7 or 8 as detected by flow cytometry. All values are mean ± SD of at least three replicates. Data were analyzed using t tests. ns = not significant ( p > 0.05).

Article Snippet: TLR7 polyclonal antibody , Bioss , Cat# bs-6601R; RRID:AB_11090775.

Techniques: Immunofluorescence, Staining, Flow Cytometry, Fluorescence

Journal: iScience

Article Title: Evidence against the role of toll-like receptors 7 and 8 in sex selection in mice, cattle, and humans

doi: 10.1016/j.isci.2025.113164

Figure Lengend Snippet: Detection of TLR7/8 on bovine X- and Y-sorted sperm (A) Immunofluorescence of TLR7 (green), TLR8 (green), acetyl α-tubulin (green) and DAPI (blue) on frozen-thawed un-sorted, X- and Y-sorted bovine sperm. (i–ii) Both TLR7 and TLR8 were stained at the acrosomal regions of the bovine sperm heads (red arrows), and tails (yellow arrows). Scale bar, 21 μm. (B, C, and E) (B) The percentages of positively stained bovine sperm for TLR7 and TLR8. Western blot analyses of TLR7 (C) and TLR8 (E) on bovine un-sorted (U), X-sorted (X) and Y-sorted (Y) sperm. TLR7 and TLR8 were resolved and blotted using non-reduced and reduced samples, respectively. (D and F) Quantification of the TLR7 (D) and TLR8 (F) bands from western blots. Data were analyzed using one-way ANOVA, ns = not significant ( p > 0.05). (G) Flow cytometry analysis of bovine sperm stained for TLR7/8 and DAPI. In controls (left panels) the sperm were probed with the secondary antibody only, which provided the cutoff (vertical line) for stained and unstained sperm. The cells were detected by the FITC-A ( x axis) and DAPI ( y axis) channels. Each dot represents one sperm. The middle and right panels represent sperm stained for TLR7 and TLR8, respectively. Nearly all sperm were stained by both antibodies. (H) The percentages of positively stained bovine sperm as detected by flow cytometry. Data were analyzed using one-way ANOVA. All values are mean ± SD of at least three replicates, ns = not significant ( p > 0.05).

Article Snippet: TLR7 polyclonal antibody , Bioss , Cat# bs-6601R; RRID:AB_11090775.

Techniques: Immunofluorescence, Staining, Western Blot, Flow Cytometry

Journal: iScience

Article Title: Evidence against the role of toll-like receptors 7 and 8 in sex selection in mice, cattle, and humans

doi: 10.1016/j.isci.2025.113164

Figure Lengend Snippet: Localization of TLR7 and TLR8 on human sperm (A) Representative images of immunofluorescence of TLR7/8 (green) and DAPI (blue) on human sperm. (i–ii) Both TLR7 and TLR8 stained human sperm tails as well as the equator regions of the heads (acrosomes, red arrows). TLR7 was also seen in the head-tail connecting apparatus (Yellow arrow). Scale bar, 22 μm. (B) The percentages of positively stained human sperm by TLR7/8 immunofluorescence. All values are mean ± SD of at least three replicates. Data were analyzed using t tests, ns = not significant ( p > 0.05). (C) Y chromosome fluorescence in situ hybridization (FISH, orange) and immunofluorescence for TLR7 and TLR8 (green) in a human sperm donor because his staining patterns and percentages were different from other donors. The inset (i) shows the details of TLR7 tail stain and the Y chromosome signal. TLR8 stained few sperm and the stain was seen on the entire sperm (ii). Immunofluorescence staining preceded FISH. Scale bar, 25 μm.

Article Snippet: TLR7 polyclonal antibody , Bioss , Cat# bs-6601R; RRID:AB_11090775.

Techniques: Immunofluorescence, Staining, Fluorescence, In Situ Hybridization

Journal: Frontiers in Immunology

Article Title: Genetic Screening for TLR7 Variants in Young and Previously Healthy Men With Severe COVID-19

doi: 10.3389/fimmu.2021.719115

Figure Lengend Snippet: (A) displays pedigrees of families 1 (patient 10) and 2 (patient 13) with segregation analysis. (B) shows schematic representation of TLR7 (HGNC ID:15631) variants reported to date in severely affected COVID-19 cases. Variants in cDNA (top) and protein (bottom). Color code: orange, variants found in the present series; blue, previously reported in van der Made ; black, previously reported in Fallerini . Shape code: circle, missense variants; square, frameshift variants. Line code: single, reported in one case; double, reported in 2 cases.

Article Snippet: Apart from a negative medium (RPMI) control, the TLR7 agonist imiquimod (imidazoquinoline compound, Invivogen) was used at a concentration of 5 μg/mL.

Techniques:

Journal: Frontiers in Immunology

Article Title: Genetic Screening for TLR7 Variants in Young and Previously Healthy Men With Severe COVID-19

doi: 10.3389/fimmu.2021.719115

Figure Lengend Snippet: Demographic, clinical, laboratory, and radiological findings of investigated patients.

Article Snippet: Apart from a negative medium (RPMI) control, the TLR7 agonist imiquimod (imidazoquinoline compound, Invivogen) was used at a concentration of 5 μg/mL.

Techniques: Imaging, Computed Tomography, Cell Counting, Coagulation, Infection

Journal: Frontiers in Immunology

Article Title: Genetic Screening for TLR7 Variants in Young and Previously Healthy Men With Severe COVID-19

doi: 10.3389/fimmu.2021.719115

Figure Lengend Snippet: Assessment of ex vivo/in vitro type I and II interferon responses in peripheral blood mononuclear cells. In all panels, patient 13 is indicated in red and the healthy controls in black. (A) shows the fold change in TLR7 mRNA expression in patient 13 and a healthy male control after stimulation for 4 hours with the TLR7 agonist imiquimod, compared to the negative medium control RPMI. (B) displays the fold change in mRNA expression of type I IFN–related genes IRF3, IRF7, IFNB1 and ISG15 induced by imiquimod as compared to RPMI. (C) shows the production of IFN-γ production after imiquimod stimulation for 7 days as compared with unstimulated control cells (RPMI) from patient 13 and two healthy male controls. The dotted line indicates the assay’s detection limit. Abbreviations: IRF3 , interferon regulatory factor 3; IRF7 , interferon regulatory factor 7; ISG15 , interferon-stimulated gene 15; and IFNB1 , interferon beta 1.

Article Snippet: Apart from a negative medium (RPMI) control, the TLR7 agonist imiquimod (imidazoquinoline compound, Invivogen) was used at a concentration of 5 μg/mL.

Techniques: Ex Vivo, In Vitro, 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: Where indicated, Enpatoran (MCE MedChem Express) or TLR7/8-IN-1 (MCE MedChem Express) was added 1 h before exposure to CL264.

Techniques: Expressing, In Vitro, Fluorescence, Microarray, Western Blot, RNA Sequencing Assay, 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: Where indicated, Enpatoran (MCE MedChem Express) or TLR7/8-IN-1 (MCE MedChem Express) was added 1 h before exposure to CL264.

Techniques: 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: Where indicated, Enpatoran (MCE MedChem Express) or TLR7/8-IN-1 (MCE MedChem Express) was added 1 h before exposure to CL264.

Techniques: Activation Assay, 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: Where indicated, Enpatoran (MCE MedChem Express) or TLR7/8-IN-1 (MCE MedChem Express) was added 1 h before exposure to CL264.

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

Journal: Cells

Article Title: TLR7 Expression Is Associated with M2 Macrophage Subset in Calcific Aortic Valve Stenosis

doi: 10.3390/cells9071710

Figure Lengend Snippet: TLR7 correlates with M2 macrophage markers in healthy, intermediate, and calcified parts of the aortic valve. ( a ) TLR7 mRNA levels were significantly increased in calcified compared with healthy and intermediate parts of the aortic valve ( n = 55). 1-way RM ANOVA with Holm-Sidak post-hoc test, **** p ≤ 0.0001. ( b – d ) Correlation analysis of TLR7 mRNA levels with mRNA levels of the VIC marker gene vimentin as well as immune cell markers in healthy ( b ), intermediate ( c ), and calcified ( d ) parts of human aortic valves ( n = 55). Pearson correlation and false discovery rate (FDR) adjusted p -values are presented.

Article Snippet: The TLR7 agonist imiquimod (IMQ, Cat. #tlrl-imq) was purchased from Invivogen (Toulouse, France), and the TLR7 inhibitor ODN20958 (Cat. #130-105-820) was purchased from Miltenyi (Bergisch Gladbach, Germany).

Techniques: Marker

Journal: Cells

Article Title: TLR7 Expression Is Associated with M2 Macrophage Subset in Calcific Aortic Valve Stenosis

doi: 10.3390/cells9071710

Figure Lengend Snippet: Gene expression of TLR7 in healthy, intermediate, and calcified parts of the aortic valve.

Article Snippet: The TLR7 agonist imiquimod (IMQ, Cat. #tlrl-imq) was purchased from Invivogen (Toulouse, France), and the TLR7 inhibitor ODN20958 (Cat. #130-105-820) was purchased from Miltenyi (Bergisch Gladbach, Germany).

Techniques: Gene Expression

Journal: Cells

Article Title: TLR7 Expression Is Associated with M2 Macrophage Subset in Calcific Aortic Valve Stenosis

doi: 10.3390/cells9071710

Figure Lengend Snippet: Co-localization of TLR7 and macrophage markers in human aortic valves, visualized by immunofluorescence staining. ( a ) TLR7 (red) is expressed in CD68 + (green) cells. ( b ) double staining of TLR7 (red) and CD206 (green). ( c ) co-localization of TLR7 (red) and CD163 (green). ( d ) TLR7 (red) is expressed in CD3 + (green) T cells. Nuclei are stained blue. Scale bars: 20 µm.

Article Snippet: The TLR7 agonist imiquimod (IMQ, Cat. #tlrl-imq) was purchased from Invivogen (Toulouse, France), and the TLR7 inhibitor ODN20958 (Cat. #130-105-820) was purchased from Miltenyi (Bergisch Gladbach, Germany).

Techniques: Immunofluorescence, Staining, Double Staining

Journal: Cells

Article Title: TLR7 Expression Is Associated with M2 Macrophage Subset in Calcific Aortic Valve Stenosis

doi: 10.3390/cells9071710

Figure Lengend Snippet: Ex vivo stimulation of human aortic valve tissue with the TLR7 agonist IMQ alters cytokine secretion. Human stenotic aortic valve tissue was stimulated ex vivo with the TLR7 ligand imiquimod (IMQ; 12.5 µg/mL). Levels of ( a ) IL-10, ( b ) TNF-α, and ( c ) GMCSF were significantly increased in the culture medium after 20 h. The effects of IMQ were significantly attenuated by the TLR7 antagonist ODN 20958 (5 µM). Data are presented as mean ± SEM. 1-way RM ANOVA with Holm-Sidak post-hoc test, * p < 0.05.

Article Snippet: The TLR7 agonist imiquimod (IMQ, Cat. #tlrl-imq) was purchased from Invivogen (Toulouse, France), and the TLR7 inhibitor ODN20958 (Cat. #130-105-820) was purchased from Miltenyi (Bergisch Gladbach, Germany).

Techniques: Ex Vivo

Journal: Cells

Article Title: TLR7 Expression Is Associated with M2 Macrophage Subset in Calcific Aortic Valve Stenosis

doi: 10.3390/cells9071710

Figure Lengend Snippet: Cytokine response from valvular interstitial cells (VICs) and primary macrophages upon in vitro stimulation with TLR7 ligand. VICs were isolated from four aortic valves derived from patients with aortic valve stenosis. Primary human macrophages were derived from monocytes isolated from blood from four healthy donors. IL-10, TNF-α, and IL-8 were measured in the culture medium after 20-h stimulation of VICs and primary macrophages, respectively, with 12.5 µg/mL IMQ. LPS was used as a positive control for VICs. ( a ) No significant change in IL-10 secretion was observed in VICs after IMQ stimulation. ( b ) TNF-α was below the detection level in VICs supernatant. ( c ) Significant increase of IL-8 upon stimulation with IMQ and LPS, respectively. 1-way ANOVA with Holm-Sidak post-hoc test, ** p < 0.01. ( d ) Increased IL-10 secretion by primary macrophages upon TLR7 activation. ( e ) No significant change in TNF-α secretion was observed in primary macrophages after IMQ stimulation. ( f ) Significant increase of IL-8 upon stimulation of primary macrophages with IMQ. Student´s paired t-test, * p < 0.05, *** p < 0.001. Data presented as mean ± SEM.

Article Snippet: The TLR7 agonist imiquimod (IMQ, Cat. #tlrl-imq) was purchased from Invivogen (Toulouse, France), and the TLR7 inhibitor ODN20958 (Cat. #130-105-820) was purchased from Miltenyi (Bergisch Gladbach, Germany).

Techniques: In Vitro, Isolation, Derivative Assay, Positive Control, Activation Assay

Journal: PLoS Genetics

Article Title: MicroRNA-3148 Modulates Allelic Expression of Toll-Like Receptor 7 Variant Associated with Systemic Lupus Erythematosus

doi: 10.1371/journal.pgen.1003336

Figure Lengend Snippet: (A) The genomic structure of the TLR7-TLR8 region and the location of all studied SNPs are indicated. (B) Association signals (−log 10 P ) are plotted against the position of each SNP (based on GRch37/hg19) in European Americans (EA), African Americans (AA), and Hispanics (HS). Genotyped and imputed SNPs are depicted with circles and triangles, respectively. The diamond identifies the TLR7 3′UTR SNP rs3853839. SNPs are highlighted using different colors according to their LD strength (r 2 ) with rs3853839. (C) A trans-ancestral meta-analysis is conducted on 40 genotyped SNPs (circles) and 14 imputed SNPs (triangles) that are shared by the three ancestries (SNPs listed in ) using fixed and random model, respectively. The dashed line represents the significance level of 5×10 −8 .

Article Snippet: For intracellular staining, PBMCs were fixed in Fixation buffer (R&D Systems) for 10 minutes at room temperature, washed twice in Permeabilization/Wash buffer (R&D Systems) and stained with PE-conjugated mouse anti-human TLR7 mAb (R&D Systems) and FITC-conjugated mouse anti-human TLR8 mAb (Imgenex) for 1 hour at room temperature.

Techniques:

Journal: PLoS Genetics

Article Title: MicroRNA-3148 Modulates Allelic Expression of Toll-Like Receptor 7 Variant Associated with Systemic Lupus Erythematosus

doi: 10.1371/journal.pgen.1003336

Figure Lengend Snippet: (A) Association of rs3853839 genotypes with TLR7/8 transcript levels in EA normal PBMCs. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (B) Association of rs3853839 genotypes with TLR7 protein levels in normal PBMCs. FACS histograms show the log MFI values plotted against the cell counts for PBMCs in individuals carrying G or C allele, compared with isotype control. Results are from one representative pair (GG or G vs. CC or C) of 7 in each gender group. MFI of TLR7 expression in PBMCs is graphically depicted. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (C) Verification of the G allele conferring elevated expression of a luciferase reporter in vitro . TLR7 3′UTR segment bearing G or C allele of rs3853839 was cloned into the psiCHECK-2 reporter vector and luciferase activity was determined after 24 hours of transfection. Relative luciferase activity is Renilla/Firefly luciferase ratio. Data show the mean ± SEM and are representative of cumulative data from four independent experiments. (D) The kinetics of the G/C allele ratio in TLR7 transcripts from PBMCs of healthy heterozygous individuals (n = 7) in the absence or presence of actinomycin D (ActD). The G/C allele ratio obtained in TLR7 transcripts was normalized to that measured from gDNA of the same sample. Data are expressed as mean ± SEM at each time point and representative of cumulative data from two independent experiments with seven healthy donors. * P <0.05. (E) Summary of the G/C allele ratio in TLR7 transcripts 4 hours after the addition of ActD or vehicle control (n = 7). Comparisons are between ActD and vehicle control cultures; P = 0.04; paired t test. FACS, Fluorescence-activated cell sorter; MFI, mean fluorescence intensity.

Article Snippet: For intracellular staining, PBMCs were fixed in Fixation buffer (R&D Systems) for 10 minutes at room temperature, washed twice in Permeabilization/Wash buffer (R&D Systems) and stained with PE-conjugated mouse anti-human TLR7 mAb (R&D Systems) and FITC-conjugated mouse anti-human TLR8 mAb (Imgenex) for 1 hour at room temperature.

Techniques: Control, Expressing, Luciferase, In Vitro, Clone Assay, Plasmid Preparation, Activity Assay, Transfection, Fluorescence

Journal: PLoS Genetics

Article Title: MicroRNA-3148 Modulates Allelic Expression of Toll-Like Receptor 7 Variant Associated with Systemic Lupus Erythematosus

doi: 10.1371/journal.pgen.1003336

Figure Lengend Snippet: (A) TargetScan's predicted miR-3148-binding site in TLR7 3′UTR. The C allele, rather than G allele of rs3853839 corresponds to the second base of this seed region. (B) Inverse correlation of miR-3148 and TLR7 transcript levels in PBMCs from 16 patients with SLE (solid circles) and 21 controls (open diamonds). (C) HEK-293 cells were cotransfected with empty reporter vector (EV), luciferase constructs driven by TLR7 3′UTR segment containing either C or G allele of rs3853839 and increasing concentrations (1, 6, 12, and 48 nM) of miR-3148 or nontarget control (NC) mimics. Luciferase activity was determined 24 hours after transfection. Normalized luciferase activity is the Renilla/Firefly ratio of miR-3148-treated reporter vector compared with the same NC-treated reporter vector. Data show the mean ± SEM and are representative of cumulative data from three independent experiments. P = 0.0003 over all miR-3148-treated C-allele vector groups, and not significant over all miR-3148-treated G-allele or empty vector groups (ANOVA test). P* = 0.02, P** <0.0001 (Student's t test) for the comparison of indicated groups.

Article Snippet: For intracellular staining, PBMCs were fixed in Fixation buffer (R&D Systems) for 10 minutes at room temperature, washed twice in Permeabilization/Wash buffer (R&D Systems) and stained with PE-conjugated mouse anti-human TLR7 mAb (R&D Systems) and FITC-conjugated mouse anti-human TLR8 mAb (Imgenex) for 1 hour at room temperature.

Techniques: Binding Assay, Plasmid Preparation, Luciferase, Construct, Control, Activity Assay, Transfection, Comparison

Journal: Cells

Article Title: TLR7 Expression Is Associated with M2 Macrophage Subset in Calcific Aortic Valve Stenosis

doi: 10.3390/cells9071710

Figure Lengend Snippet: TLR7 correlates with M2 macrophage markers in healthy, intermediate, and calcified parts of the aortic valve. ( a ) TLR7 mRNA levels were significantly increased in calcified compared with healthy and intermediate parts of the aortic valve ( n = 55). 1-way RM ANOVA with Holm-Sidak post-hoc test, **** p ≤ 0.0001. ( b – d ) Correlation analysis of TLR7 mRNA levels with mRNA levels of the VIC marker gene vimentin as well as immune cell markers in healthy ( b ), intermediate ( c ), and calcified ( d ) parts of human aortic valves ( n = 55). Pearson correlation and false discovery rate (FDR) adjusted p -values are presented.

Article Snippet: Primary antibodies were purchased from Abcam (Cambridge, UK; Anti-CD163 antibody (Cat. #ab74604)), Agilent (Santa Clara, CA, USA; Anti-CD68 (Dako Omnis) (Cat. #M0876)), R&D systems (Minneapolis, MN, USA; Human MR/CD206 Antibody (Cat. #AF2534)), Novus Biologicals (Cambridge, UK; Anti-TLR7 antibody (Cat. #NBP2-24906)), and Biocare Medical (Pacheco, CA, USA; human Anti-CD3 antibody (Cat.#PM110)) respectively.

Techniques: Marker

Journal: Cells

Article Title: TLR7 Expression Is Associated with M2 Macrophage Subset in Calcific Aortic Valve Stenosis

doi: 10.3390/cells9071710

Figure Lengend Snippet: Gene expression of TLR7 in healthy, intermediate, and calcified parts of the aortic valve.

Article Snippet: Primary antibodies were purchased from Abcam (Cambridge, UK; Anti-CD163 antibody (Cat. #ab74604)), Agilent (Santa Clara, CA, USA; Anti-CD68 (Dako Omnis) (Cat. #M0876)), R&D systems (Minneapolis, MN, USA; Human MR/CD206 Antibody (Cat. #AF2534)), Novus Biologicals (Cambridge, UK; Anti-TLR7 antibody (Cat. #NBP2-24906)), and Biocare Medical (Pacheco, CA, USA; human Anti-CD3 antibody (Cat.#PM110)) respectively.

Techniques: Expressing

Journal: Cells

Article Title: TLR7 Expression Is Associated with M2 Macrophage Subset in Calcific Aortic Valve Stenosis

doi: 10.3390/cells9071710

Figure Lengend Snippet: Co-localization of TLR7 and macrophage markers in human aortic valves, visualized by immunofluorescence staining. ( a ) TLR7 (red) is expressed in CD68 + (green) cells. ( b ) double staining of TLR7 (red) and CD206 (green). ( c ) co-localization of TLR7 (red) and CD163 (green). ( d ) TLR7 (red) is expressed in CD3 + (green) T cells. Nuclei are stained blue. Scale bars: 20 µm.

Article Snippet: Primary antibodies were purchased from Abcam (Cambridge, UK; Anti-CD163 antibody (Cat. #ab74604)), Agilent (Santa Clara, CA, USA; Anti-CD68 (Dako Omnis) (Cat. #M0876)), R&D systems (Minneapolis, MN, USA; Human MR/CD206 Antibody (Cat. #AF2534)), Novus Biologicals (Cambridge, UK; Anti-TLR7 antibody (Cat. #NBP2-24906)), and Biocare Medical (Pacheco, CA, USA; human Anti-CD3 antibody (Cat.#PM110)) respectively.

Techniques: Immunofluorescence, Staining, Double Staining

Journal: Cells

Article Title: TLR7 Expression Is Associated with M2 Macrophage Subset in Calcific Aortic Valve Stenosis

doi: 10.3390/cells9071710

Figure Lengend Snippet: Ex vivo stimulation of human aortic valve tissue with the TLR7 agonist IMQ alters cytokine secretion. Human stenotic aortic valve tissue was stimulated ex vivo with the TLR7 ligand imiquimod (IMQ; 12.5 µg/mL). Levels of ( a ) IL-10, ( b ) TNF-α, and ( c ) GMCSF were significantly increased in the culture medium after 20 h. The effects of IMQ were significantly attenuated by the TLR7 antagonist ODN 20958 (5 µM). Data are presented as mean ± SEM. 1-way RM ANOVA with Holm-Sidak post-hoc test, * p < 0.05.

Article Snippet: Primary antibodies were purchased from Abcam (Cambridge, UK; Anti-CD163 antibody (Cat. #ab74604)), Agilent (Santa Clara, CA, USA; Anti-CD68 (Dako Omnis) (Cat. #M0876)), R&D systems (Minneapolis, MN, USA; Human MR/CD206 Antibody (Cat. #AF2534)), Novus Biologicals (Cambridge, UK; Anti-TLR7 antibody (Cat. #NBP2-24906)), and Biocare Medical (Pacheco, CA, USA; human Anti-CD3 antibody (Cat.#PM110)) respectively.

Techniques: Ex Vivo

Journal: Cells

Article Title: TLR7 Expression Is Associated with M2 Macrophage Subset in Calcific Aortic Valve Stenosis

doi: 10.3390/cells9071710

Figure Lengend Snippet: Cytokine response from valvular interstitial cells (VICs) and primary macrophages upon in vitro stimulation with TLR7 ligand. VICs were isolated from four aortic valves derived from patients with aortic valve stenosis. Primary human macrophages were derived from monocytes isolated from blood from four healthy donors. IL-10, TNF-α, and IL-8 were measured in the culture medium after 20-h stimulation of VICs and primary macrophages, respectively, with 12.5 µg/mL IMQ. LPS was used as a positive control for VICs. ( a ) No significant change in IL-10 secretion was observed in VICs after IMQ stimulation. ( b ) TNF-α was below the detection level in VICs supernatant. ( c ) Significant increase of IL-8 upon stimulation with IMQ and LPS, respectively. 1-way ANOVA with Holm-Sidak post-hoc test, ** p < 0.01. ( d ) Increased IL-10 secretion by primary macrophages upon TLR7 activation. ( e ) No significant change in TNF-α secretion was observed in primary macrophages after IMQ stimulation. ( f ) Significant increase of IL-8 upon stimulation of primary macrophages with IMQ. Student´s paired t-test, * p < 0.05, *** p < 0.001. Data presented as mean ± SEM.

Article Snippet: Primary antibodies were purchased from Abcam (Cambridge, UK; Anti-CD163 antibody (Cat. #ab74604)), Agilent (Santa Clara, CA, USA; Anti-CD68 (Dako Omnis) (Cat. #M0876)), R&D systems (Minneapolis, MN, USA; Human MR/CD206 Antibody (Cat. #AF2534)), Novus Biologicals (Cambridge, UK; Anti-TLR7 antibody (Cat. #NBP2-24906)), and Biocare Medical (Pacheco, CA, USA; human Anti-CD3 antibody (Cat.#PM110)) respectively.

Techniques: In Vitro, Isolation, Derivative Assay, Positive Control, Activation Assay

Journal: BMC Pregnancy and Childbirth

Article Title: Differential immunophenotype of circulating monocytes from pregnant women in response to viral ligands

doi: 10.1186/s12884-023-05562-0

Figure Lengend Snippet: Monocyte response to TLR7/TLR8 stimulation. A Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of pregnant ( n = 20) and non-pregnant ( n = 20) women and stimulated with R848 (TLR7/TLR8 agonist) or Gardiquimod (TLR7 agonist) for 24 h. Flow cytometry was performed to phenotype monocytes. B Flow cytometry gating strategy for phenotyping of monocyte subsets after in vitro stimulation with viral ligands. Viable monocytes were gated as live CD14 + cells from PBMCs. The expression levels of CD16 and CD14 were used to gate monocyte subsets as follows: classical (CD14 hi CD16 − ); intermediate (CD14 hi CD16 + ); non-classical (CD14 lo CD16 + ), and CD14 lo CD16 − . C Proportions of monocyte subsets in pregnant (red) and non-pregnant (blue) women with and without R848 stimulation. D Heatmap representation of the differences in proportions of monocytes subsets from pregnant (red symbols) and non-pregnant (blue symbols) following R848 stimulation. Asterisks indicate statistically significant differences between the indicated groups. E - N Frequencies of ( E ) classical monocytes, ( F ) intermediate monocytes, ( G ) non-classical monocytes, ( H ) CD14 lo CD16 − monocytes, ( I ) CD147 + monocytes, ( J ) CD162 + monocytes, ( K ) CCR5 + CCR2 + monocytes, ( L ) CCR5 − CCR2 + monocytes, ( M ) CCR5 − CCR2 − monocytes, and ( N ) CCR5 + CCR2 − monocytes in pregnant (red) and non-pregnant (blue) women following R848 stimulation (solid circles) or control (open circles). * p < 0.05; ** p < 0.01; *** p < 0.001. ( +) Stimulated; (-) Control

Article Snippet: For viral ligand stimulation, PBMCs were individually incubated with 2.5 µg/mL R848 (TLR7/8-based adjuvant; Cat# vac-r848; InvivoGen, San Diego, CA, USA), 1 µM Gardiquimod (TLR7 ligand; Cat# tlrl-gdqs; InvivoGen), 10 µg/mL Poly(I:C) (HMW) VacciGrade™ (TLR3-based adjuvant; Cat# vac-pic; InvivoGen), 50 µg/mL Poly(I:C) (HMW) LyoVec™ (RIG-I/MDA-5 ligand; Cat# tlrl-piclv; InvivoGen), and 2 µg/mL ODN 2216 (TLR9 ligand; Cat# tlrl-2216; InvivoGen) at 37 °C with 5% CO 2 for 24 h with the addition of protein transport inhibitor cocktail (Cat# 00-4980-03; ThermoFisher Scientific) for the last 4 h of incubation.

Techniques: Isolation, Flow Cytometry, In Vitro, Expressing, Control

Journal: BMC Pregnancy and Childbirth

Article Title: Differential immunophenotype of circulating monocytes from pregnant women in response to viral ligands

doi: 10.1186/s12884-023-05562-0

Figure Lengend Snippet: Monocyte response to TLR7 stimulation. A Proportions of monocyte subsets in pregnant (red) and non-pregnant women (blue) with and without Gardiquimod stimulation (TLR7 agonist). B Heatmap representation of the differences in proportions of monocyte subsets from pregnant (red symbols) and non-pregnant (blue symbols) women following Gardiquimod stimulation. Asterisks indicate statistically significant differences between the indicated groups. C - F Frequencies of ( C ) classical monocytes (CD14 hi CD16 − ), ( D ) intermediate monocytes (CD14 hi CD16 + ), ( E ) non-classical monocytes (CD14 lo CD16 + ), ( F ) CD14 lo CD16 − monocytes in pregnant (red) and non-pregnant (blue) women following Gardiquimod stimulation (solid circles) or control (open circles). * p < 0.05; ** p < 0.01. ( +) Stimulated; (-) Control

Article Snippet: For viral ligand stimulation, PBMCs were individually incubated with 2.5 µg/mL R848 (TLR7/8-based adjuvant; Cat# vac-r848; InvivoGen, San Diego, CA, USA), 1 µM Gardiquimod (TLR7 ligand; Cat# tlrl-gdqs; InvivoGen), 10 µg/mL Poly(I:C) (HMW) VacciGrade™ (TLR3-based adjuvant; Cat# vac-pic; InvivoGen), 50 µg/mL Poly(I:C) (HMW) LyoVec™ (RIG-I/MDA-5 ligand; Cat# tlrl-piclv; InvivoGen), and 2 µg/mL ODN 2216 (TLR9 ligand; Cat# tlrl-2216; InvivoGen) at 37 °C with 5% CO 2 for 24 h with the addition of protein transport inhibitor cocktail (Cat# 00-4980-03; ThermoFisher Scientific) for the last 4 h of incubation.

Techniques: Control

Journal: BMC Pregnancy and Childbirth

Article Title: Differential immunophenotype of circulating monocytes from pregnant women in response to viral ligands

doi: 10.1186/s12884-023-05562-0

Figure Lengend Snippet: Interferon production by peripheral blood mononuclear cells (PBMCs) upon TLR7/TLR8 or TLR3 stimulation. A Peripheral blood samples were collected from pregnant ( n = 20, indicated in red) and non-pregnant ( n = 20, indicated in blue) women to isolate PBMCs for in vitro stimulation with R848 or Poly(I:C) (HMW) VacciGrade™ [Poly(I:C)]. Type-I (IFN-α2a, -β), Type-II (IFN-γ) and Type-III (IL-29/IFN-λ1) interferon concentrations were then determined in culture supernatants. B - E Log 10 -transformed concentrations of ( B ) IFN-α2a, ( C ) IFN-β, ( D ) IFN-γ, and ( E ) IL-29/IFN-λ1 in culture supernatants of PBMCs from pregnant (red symbols) and non-pregnant (blue symbols) women in response to R848 (solid circles) or control (open circles). F - I Log 10 -transformed concentrations of ( F ) IFN-α2a, ( G ) IFN-β, ( H ) IFN-γ, and ( I ) IL-29/IFN-λ1 in culture supernatants of PBMCs from pregnant (red symbols) and non-pregnant (blue symbols) women in response to Poly(I:C) (solid circles) or control (open circles). Dotted lines indicate the detection limit of each analyte. ** p < 0.01, *** p < 0.001. ( +) Stimulated, (-) Control

Article Snippet: For viral ligand stimulation, PBMCs were individually incubated with 2.5 µg/mL R848 (TLR7/8-based adjuvant; Cat# vac-r848; InvivoGen, San Diego, CA, USA), 1 µM Gardiquimod (TLR7 ligand; Cat# tlrl-gdqs; InvivoGen), 10 µg/mL Poly(I:C) (HMW) VacciGrade™ (TLR3-based adjuvant; Cat# vac-pic; InvivoGen), 50 µg/mL Poly(I:C) (HMW) LyoVec™ (RIG-I/MDA-5 ligand; Cat# tlrl-piclv; InvivoGen), and 2 µg/mL ODN 2216 (TLR9 ligand; Cat# tlrl-2216; InvivoGen) at 37 °C with 5% CO 2 for 24 h with the addition of protein transport inhibitor cocktail (Cat# 00-4980-03; ThermoFisher Scientific) for the last 4 h of incubation.

Techniques: In Vitro, Transformation Assay, Control