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: Nature Communications

Article Title: DPEP2 suppresses hyperinflammation via metabolic reprogramming of macrophages in sepsis

doi: 10.1038/s41467-026-70466-4

Figure Lengend Snippet: a mRNA levels of Dpep2 in RAW264.7 cells were examined after stimulation with Pam3Csk4 (TLR1/2 ligand), LPS (TLR4 ligand), R848 (TLR7/8 ligand) and poly (I:C) (TLR3 ligand) for 12 h ( n = 3 biological replicates). b , c mRNA expressions of DPEP2 in human monocytes ( b ) or bone marrow-derived macrophages (BMDM) ( c ) were examined after LPS stimulation for 12 h with different doses ( n = 3 biological replicates). d mRNA expressions of Dpep2 in RAW264.7 cells were examined after LPS stimulation for 6 or 12 h with different doses ( n = 3 biological replicates). e Protein levels of DPEP2 in RAW264.7 cells were examined after LPS stimulation for 24 h with different doses ( n = 3 biological replicates). f – g RAW264.7 cells with Dpep2 deficiency (sh Dpep2 RAW264.7 cells, n = 4 biological replicates) and control RAW264.7 cells (shNT RAW264.7 cells, n = 3 biological replicates) were obtained for RNA-seq analysis; f Volcano plot showing results from differential expression analysis. Genes with P-adjusted value < 0.05 are highlighted in red or blue; g Bar plot showing the GSEA results in sh Dpep2 vs shNT RAW264.7 cells. h – k Levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF) in sh Dpep2 vs shNT BMDMs and Dpep2 knockout ( Dpep2 KO) vs wild type (WT) mice derived BMDMs were analyzed ( n = 3 biological replicates). l, m IL-1β, IL-6 and TNF secretion of sh Dpep2 vs shNT RAW264.7 cells and Dpep2 KO vs WT mice derived BMDMs were analyzed at 24 h after LPS stimulation ( n = 3 biological replicates). n - o RAW264.7 cells and BMDMs overexpressing Dpep2 (DPEP2) and relatively control vector (Vector) cells were constructed. Levels of IL-1β, IL-6, and TNF secreted by DPEP2 cells vs Vector cells were analyzed at 24 h after LPS stimulation ( n = 3 biological replicates). Data represent the mean ± standard deviation. ns, not significant. One-way ANOVA followed by the Bonferroni multiple comparisons was used in ( a – c, e ), two-way repeated measures ANOVA with Tukey’s multiple comparisons test was used in ( d ), two-sided Wilcoxon rank-sum test was used in ( f ) and two-sided unpaired Student’s t test was used in ( h–o ). NES, n o rmalized enrichment score.

Article Snippet: RAW264.7 cells were treated with TLR1/2 ligands (1 μg/mL Pam3Csk4; HY-P1180A, MedChemExpress, Monmouth Junction, NJ, USA), TLR7/8 ligands (1 μg/mL R848; HY-13740, MedChemExpress) and TLR3 ligands (1 μg/mL poly(I:C); HY-107202, MedChemExpress) for 12 h. RAW264.7 cells, human monocytes, and BMDMs were stimulated with LPS (L4391, Sigma) to establish an in vitro inflammatory model. RAW264.7 cells were treated with LTD4 (1 μM; 20310, Cayman, Ann Arbor, MI, USA), LTE4 (1 μM; 20410, Cayman) or PGE2 (1 μM; HY-101952, MedChemExpress) in vitro.

Techniques: Derivative Assay, Control, RNA Sequencing, Quantitative Proteomics, Knock-Out, Plasmid Preparation, Construct, Standard Deviation

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: 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: 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: Frontiers in Immunology

Article Title: Cyclophilin–CD147 interaction enables SARS-CoV-2 infection of human monocytes and their activation via Toll-like receptors 7 and 8

doi: 10.3389/fimmu.2025.1460089

Figure Lengend Snippet: SARS-CoV-2 activates monocytes via endosomal ssRNA-sensing TLRs. (A) MACS-sorted human blood monocytes or THP-1 NF-κB::eGFP reporter cells were infected with SARS-CoV-2 (MOI 2), or treated with the respective mock supernatant, or left untreated, for 24 h. Expression of TLR7 , TLR8 and TLR3 was measured by RT-qPCR (n = 3-4). (B) MACS-sorted monocytes were infected with SARS-CoV-2 at MOI 2, or the respective mock supernatant, or left untreated for 24 h, and if indicated, pretreated with the TLR7/8 antagonist enpatoran (1 μM). As controls for proper TLR7/8 stimulation, monocytes were in parallel treated with the TLR7/8 agonist resiquimod (2.5 μg/ml) for 24 h and pretreated or not with enpatoran (1 μM). TNF, IL1B, IL6 , CXCL8, IL10 and IL15 mRNA levels were determined using RT-qPCR (n = 5 donors). (C) MACS-sorted monocytes were either mock-transfected or transfected with the in vitro -transcribed 4.5 kb fragment of SARS-CoV-2 RNA and were pretreated (or not) with enpatoran (1 μM). As controls, cells were also treated with resiquimod (2.5 μg/ml) in the presence or absence of enpatoran. After 24 h, TNF, IL1B, IL6 , CXCL8, IL10 and IL15 mRNA levels were quantified by RT-qPCR (n = 4 donors). Data in (A–C) are represented as mean ± SEM and statistical significance was assessed using two-way (A) or one-way (C) ANOVA with Tukey’s post-hoc test, or using two independent one-way ANOVA tests with Tukey’s post-hoc test (B) , where their significances are depicted in normal or bold letters, respectively.

Article Snippet: TLRs were inhibited by an anti-TLR2 monoclonal antibody (mAb) or treated with an isotype control mAb (both Miltenyi Biotec, and used at final 10 μg/ml), or by TLR7/8 inhibitor enpatoran (MedChemExpress; final 1 μM) 1 h before SARS-CoV-2 infection.

Techniques: Infection, Expressing, Quantitative RT-PCR, Transfection, In Vitro

Journal: bioRxiv

Article Title: Long noncoding RNA AL109754.1 Regulates Myeloid Dendritic Cell Differentiation and Potentiates TLR signaling

doi: 10.1101/2023.03.01.530613

Figure Lengend Snippet: In vitro human monocyte-derived DC were transfected with scramble or si-AL109754.1. After 48h, cells were challenged with TLR2, TLR4, TLR5, TLR7 or TLR9 agonists. ( A ) Effect of lncRNA AL109754.1 knockdown on NFkB phosphorylation, IRF3, and IRF7 using phosphoflow. The half overlaid histograms in left panel were generated in Flowjo_v10.8.1 to elucidate the comparison phosphorylated species in control vs si-AL109754.1 transfected cells. After 48h post transfections all the cells were fixed with Cytofix Buffer (BD Biosciences), permeabilized with methanol and stained with various phospho-antibodies for TFs-associated with the TLR signaling. ( B ) Representative scatter plots show the phosphorylated percentage of downstream TLR molecules generated in Flowjo_v10.8.1. ( C ) Bar graph showing average percentage of cells containing phosohorylated TLR specific downstream signaling molecules. Each bar shows mean ±SD. Students t-test were used to calculate p-values and p <0.05 was considered significant. *p<0.05, **p<0.01, ***p<0.001 .

Article Snippet: For TLR staining experiments, cells were stained with TLR1 (BV421 conjugated Mouse Anti-Human CD281, clone: GD2.F4, BD Biosciences), TLR2 (FITC conjugated anti-human CD282 Antibody, clone: W15145C, BioLegend), TLR3 (Brilliant Violet 711TM conjugated anti-human CD283, clone: TLR-104, BioLegend), TLR4 (PE conjugated anti-human CD284, clone: HTA125, BioLegend), TLR5 (Alexa Fluor® 488 conjugated anti-human CD285 Antibody, clone: S16021I, BioLegend), TLR6 (PE conjugated anti-human CD286 Antibody, clone: TLR6.127, BioLegend), TLR7 (Alexa Fluor® 700-conjugated Antibody, clone: 533707, R&D Systems), TLR8 (PE anti-human CD288 Antibody, clone: S16018A, BioLegend) and TLR9 (APC conjugated anti-human CD289 Antibody, clone: S16013D, BioLegend) antibodies.

Techniques: In Vitro, Derivative Assay, Transfection, Knockdown, Phospho-proteomics, Generated, Comparison, Control, Staining

Journal: bioRxiv

Article Title: Long noncoding RNA AL109754.1 Regulates Myeloid Dendritic Cell Differentiation and Potentiates TLR signaling

doi: 10.1101/2023.03.01.530613

Figure Lengend Snippet: Scramble or si-AL109754.1 transfected DCs were challenged with TLR4 (concentration), TLR5 (concentration), and TLR7 (concentration) agonists. After 18h post-challenge, total RNA was isolated using miRNeasy Kit (Qiagen). cDNA was synthesized from 500 ng total RNA and the expression of NFκB pathway genes was assessed by custom PCR array containing 88 gene primers. The top of the heat map shows the sample identifiers. Heat maps showing differentially expressed genes in AL109754.1 knockdown DC (n=3/group) challenged with (A) TLR4 agonist, (B) TLR5 agonist and (C) TLR8 agonist. The data was analyzed using the Qiagen GeneGlobe Data Analysis Center (Source: https://www.qiagen.com/us/shop/genesand-pathways/data-analysis-center-overview-page/ ). B2M was used as housekeeping to normalize the expression levels. The fold change was used to analyze the changes NFκB pathway related genes in DCs challenged with TLR agonists (scramble vs si-AL109754.1; p<0.05 ). (B) Venn diagram showing the overview of differential gene expression of NFκB pathway upon TLR stimulation in AL109754.1 knockdown DCs.

Article Snippet: For TLR staining experiments, cells were stained with TLR1 (BV421 conjugated Mouse Anti-Human CD281, clone: GD2.F4, BD Biosciences), TLR2 (FITC conjugated anti-human CD282 Antibody, clone: W15145C, BioLegend), TLR3 (Brilliant Violet 711TM conjugated anti-human CD283, clone: TLR-104, BioLegend), TLR4 (PE conjugated anti-human CD284, clone: HTA125, BioLegend), TLR5 (Alexa Fluor® 488 conjugated anti-human CD285 Antibody, clone: S16021I, BioLegend), TLR6 (PE conjugated anti-human CD286 Antibody, clone: TLR6.127, BioLegend), TLR7 (Alexa Fluor® 700-conjugated Antibody, clone: 533707, R&D Systems), TLR8 (PE anti-human CD288 Antibody, clone: S16018A, BioLegend) and TLR9 (APC conjugated anti-human CD289 Antibody, clone: S16013D, BioLegend) antibodies.

Techniques: Transfection, Concentration Assay, Isolation, Synthesized, Expressing, Knockdown, Gene Expression

Journal: Frontiers in Immunology

Article Title: UNC93B1 Is Widely Expressed in the Murine CNS and Is Required for Neuroinflammation and Neuronal Injury Induced by MicroRNA let-7b

doi: 10.3389/fimmu.2021.715774

Figure Lengend Snippet: Neuroinflammatory response and neuronal injury in response to extracellular let-7b are dependent on UNC93B1 in vitro . Microglia derived from C57BL/6 (wild-type, WT) and Unc93b1 -/- mice were incubated with various doses of let-7b oligoribonucleotide, as indicated, for 12 h (A) , or with 10 µg/ml let-7b for various durations, as indicated (B) . Mutant oligoribonucleotide (20 µg/ml, 12 h) and the transfection agent LyoVec served as negative control. LPS (100 ng/ml, 12 h) was used as positive control for microglial TNF-α release through TLR4 activation, while loxoribine (1 mM, 12 h) served as control for TLR7-dependent TNF-α release. Subsequently, supernatants were analyzed by TNF-α ELISA. Results are presented as mean ± SD. n = 4; n.d., not detectable. (C–E) WT neurons co-cultured with either WT or Unc93b1 -/- microglia were treated with various let-7b doses ( C, D : 10 µg/ml for 72 h; E : as indicated for 5 d). Mutant oligoribonucleotide (5 µg/ml) served as negative control, LPS (100 ng/ml) served as positive control for microglial activation, and loxoribine (10 mM) served as positive control for TLR7-dependent microglial activation. Subsequently, co-cultures were immunolabeled with Iba1 (C) and NeuN (D) antibodies to mark microglia and neurons, respectively. Scale bar, 50 µm. (E) Quantification of NeuN-positive cells in co-cultures treated as described above. Untreated control was set to 100%. For each condition, experiments were performed in duplicates. At least 3 independent experiments were performed. Data are expressed as mean ± SEM. Kruskal-Wallis test was used to determine global significance over all conditions ( p = 0.0406 over WT and p = 0.0897 over Unc93b1 -/- ). P values of relevant groups (indicated condition vs. unstimulated control, ctrl) were determined by Dunn’s multiple comparison test. P values between the respective groups of WT vs. Unc93b1 -/- (in brackets) were assessed by unpaired Student’s t test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The following primary antibodies were used: anti-NeuN (1:1000 in vitro , 1:500 for sections, cat. #ABN78), anti-Neurofilament (1:1000 in vitro , 1:500 for sections, clone DA2), anti-microtubule-associated protein 2 (MAP-2, 1:1000, clone AP20), anti-GFAP (1:1000 in vitro , 1:500 for sections, clone AB5541), anti-APC (1:300, OP80), anti-active caspase-3 (1:450, clone AB3623, all obtained from Merck Millipore, Burlington, MA, USA), anti-Iba1 (1:1000 in vitro , 1:500 for sections; cat. #019-19741, Wako, Neuss, Germany), anti-CD11b (1:500, M1/70, Thermofisher Scientific, cat. #14-0112-82, Waltham, MA, USA), and anti-TLR7 (1:500; Novus Biologicals cat. #NBP2-27332, Centennial, CO, USA.

Techniques: In Vitro, Derivative Assay, Incubation, Mutagenesis, Transfection, Negative Control, Positive Control, Activation Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Immunolabeling, Comparison

Journal: Frontiers in Immunology

Article Title: UNC93B1 Is Widely Expressed in the Murine CNS and Is Required for Neuroinflammation and Neuronal Injury Induced by MicroRNA let-7b

doi: 10.3389/fimmu.2021.715774

Figure Lengend Snippet: UNC93B1 is required for let-7b -induced cell-autonomous neuronal apoptosis in vitro . (A, B) Enriched cortical neurons isolated from C57BL/6 (wild-type, WT) and Unc93b1 -/- mice were exposed to 10 µg/ml let-7b for 72 h or were left untreated (control). Mutant oligoribonucleotide (5 µg/ml) served as negative control. LPS (100 ng/ml) was used to indicate functionally relevant microglia cell numbers in the enriched neuronal cell cultures. Loxoribine (10 mM) served as positive control for TLR7-dependent neuronal injury. Subsequently, cells were immunolabeled using Neurofilament (A) and NeuN (B) antibodies to assess axonal damage and neuronal viability, respectively. (C, D) Cell cultures derived from WT and Unc93b1 -/- mice described above were incubated with various doses of let-7b , as indicated, for 72 h (C) or treated with 5 µg/ml let-7b for different time periods, as indicated (D) . (E–H) WT cortical neurons were incubated with let-7b (5 µg/ml) for various time periods, as indicated, and were subsequently analyzed by (E) TUNEL assay and (G) immunostaining using active caspase-3 antibody. DAPI staining marked all present cells. Subsequently, TUNEL-positive and caspase-3-positive cells were quantified and normalized against DAPI-positive cells ( F and H , respectively). Mutant oligoribonucleotide served as negative control, while loxoribine and LPS were used as positive control. (C–H) At least three independent experiments were performed. Data are expressed as mean ± SEM. Kruskal-Wallis test was used to determine global significance over all conditions [ (C) p = 0.0042 over WT and p = 0.9557 over Unc93b1 -/- ; (D) p = 0.0036 over WT and p = 0.9971 over Unc93b1 -/- ; (F) p < 0.0001 over WT and p = 0.3514 over Unc93b1 -/- ; (H) p = 0.0010 over WT and p = 0.0036 over Unc93b1 -/- ]. P values of relevant groups (indicated condition vs. unstimulated control, ctrl) were determined by Dunn’s multiple comparison test. P values between respective groups of WT vs. Unc93b1 -/- (in brackets) were assessed by unpaired Student’s t test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Scale bar, 50 µm.

Article Snippet: The following primary antibodies were used: anti-NeuN (1:1000 in vitro , 1:500 for sections, cat. #ABN78), anti-Neurofilament (1:1000 in vitro , 1:500 for sections, clone DA2), anti-microtubule-associated protein 2 (MAP-2, 1:1000, clone AP20), anti-GFAP (1:1000 in vitro , 1:500 for sections, clone AB5541), anti-APC (1:300, OP80), anti-active caspase-3 (1:450, clone AB3623, all obtained from Merck Millipore, Burlington, MA, USA), anti-Iba1 (1:1000 in vitro , 1:500 for sections; cat. #019-19741, Wako, Neuss, Germany), anti-CD11b (1:500, M1/70, Thermofisher Scientific, cat. #14-0112-82, Waltham, MA, USA), and anti-TLR7 (1:500; Novus Biologicals cat. #NBP2-27332, Centennial, CO, USA.

Techniques: In Vitro, Isolation, Mutagenesis, Negative Control, Positive Control, Immunolabeling, Derivative Assay, Incubation, TUNEL Assay, Immunostaining, Staining, Comparison

Journal: Molecules and Cells

Article Title: STING Negatively Regulates Double-Stranded DNA-Activated JAK1-STAT1 Signaling via SHP-1/2 in B Cells

doi: 10.14348/molcells.2015.2359

Figure Lengend Snippet: Low-expression of STING in B cells from patients with SLE and MRL/ lpr mice. (A) FACS analysis of STING expression in B cells from patients with SLE (n = 8) and healthy donors (n = 8). (B) qPCR analysis of the expression of STING in B cells from MRL/ lpr mice (n = 9) and wild-type mice (n = 10). (C) B cells were isolated from spleen of MRL/ lpr mice (n = 6) and wild-type mice (n = 6). Western blot analysis of the phosphorylation levels of SHP-1/2 and JAK1. (D) BJAB cells were transfected with poly(dA:dT) for 8 h,16 h and 24 h. The expression of STING was analyzed by qPCR. (E) Isolated human CD19 + B cells were stimulated with TLR7 ligand R848 (1 μg/ml), TLR9 ligand CpG2006S (0.5 μM), human anti-IgM (10 μg/ml) and human IFN-α (1,000 U/ml) for 8 h and 16 h. The expression of STING was detected by qPCR. (F) Isolated murine splenic B cells were stimulated with TLR7 ligand R848 (1 μg/ml), TLR9 ligand CpG1826 (0.5 μM), mouse anti-IgM (10 μg/ml) and mouse IFN-α (1,000 U/ml) for 8 h and 16 h. The expression of STING was detected by qPCR. (G) Proposed regulatory pathway activated by dsDNA in B cells. The data shown represent the means of three independent experiments, and the error bars represent the s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001; ns denotes p > 0.05.

Article Snippet: For in vitro experiments, isolated human CD19+ B cells were cultured in RPMI 1640 medium containing 10% FBS and stimulated with TLR7 ligand R848 (1 μg/ml, Enzo Life Sciences International), TLR9 ligand CpG-2006S (0.3 μM, Invitrogen), AffiniPure F(ab’) 2 Fragment Goat Anti-human IgM (10 μg/ml, Jackson ImmunoResearch Laboratories) or human IFN-α (1000 U/ml, Millipore).

Techniques: Expressing, Isolation, Western Blot, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Toll-like Receptor 7 (TLR7) Is Expressed in Adipocytes and the Pharmacological TLR7 Agonist Imiquimod and Adipocyte-Derived Cell-Free Nucleic Acids (cfDNA) Regulate Adipocyte Function

doi: 10.3390/ijms23158475

Figure Lengend Snippet: Imiquimod reduces both adiponectin and resistin mRNA levels in 3T3-L1 adipocytes in vitro. Mature 3T3-L1 adipocytes ( n = 11–12 wells each) were treated with different doses of TLR7 agonist imiquimod (IMQ) for 18 h as described in the Materials and Methods section. After isolation of mRNA applying the RNeasy ® Mini Kit (Qiagen, Hilden, Germany), gene expression levels of murine adiponectin and resistin were quantified by reverse transcription and real-time PCR (RT-PCR) of the corresponding cDNA and normalized to GAPDH, as described in the Methods section. Imiquimod reduced both ( a ) adiponectin and ( b ) resistin mRNA levels.

Article Snippet: After isolation of mRNA applying the RNeasy ® Mini Kit (Qiagen, Hilden, Germany), the gene expression levels of murine TLR7, TLR9, adiponectin, resistin, CD45, glucose transporter 1 (GLUT1) and glucose transporter 4 (GLUT4) and of human TLR7 and TLR9 were quantified by reverse transcription and real-time PCR (RT-PCR) of the corresponding cDNA.

Techniques: In Vitro, Isolation, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Toll-like Receptor 7 (TLR7) Is Expressed in Adipocytes and the Pharmacological TLR7 Agonist Imiquimod and Adipocyte-Derived Cell-Free Nucleic Acids (cfDNA) Regulate Adipocyte Function

doi: 10.3390/ijms23158475

Figure Lengend Snippet: Cell-free nucleic acids (cfDNA) reduce both adiponectin and resistin mRNA levels in 3T3-L1 adipocytes in vitro. Mature 3T3-L1 adipocytes ( n = 17–18 wells each) were treated with different doses of cfDNA (100 ng/mL, 1 μg/mL) generated from TNF-exposed 3T3-L1 adipocytes as described in the Materials and Methods section for 18 h. After isolation of mRNA applying the RNeasy ® Mini Kit (Qiagen, Hilden, Germany), gene expression levels of murine adiponectin and resistin were quantified by reverse transcription and real-time PCR (RT-PCR) of the corresponding cDNA and normalized to GAPDH, as described in the Materials and Methods section. cfDNA reduced both ( a ) adiponectin and ( b ) resistin mRNA levels.

Article Snippet: After isolation of mRNA applying the RNeasy ® Mini Kit (Qiagen, Hilden, Germany), the gene expression levels of murine TLR7, TLR9, adiponectin, resistin, CD45, glucose transporter 1 (GLUT1) and glucose transporter 4 (GLUT4) and of human TLR7 and TLR9 were quantified by reverse transcription and real-time PCR (RT-PCR) of the corresponding cDNA.

Techniques: In Vitro, Generated, Isolation, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Toll-like Receptor 7 (TLR7) Is Expressed in Adipocytes and the Pharmacological TLR7 Agonist Imiquimod and Adipocyte-Derived Cell-Free Nucleic Acids (cfDNA) Regulate Adipocyte Function

doi: 10.3390/ijms23158475

Figure Lengend Snippet: Imiquimod reduces adiponectin while increasing MCP1/CCL2 mRNA levels in 3T3-L1 adipocytes in co-culture with murine J774A.1 monocytes in vitro. Mature 3T3-L1 adipocytes were treated with IMQ (+IMQ) separately (3T3) and in co-culture (3T3 + J774) with J774A.1 monocytes as described in the Methods section for 18 h ( n = 7–12 wells each). After isolation of mRNA from the adipocytes applying the RNeasy ® Mini Kit (Qiagen, Hilden, Germany), gene expression levels of murine adiponectin, resistin and MCP1/CCL2 were quantified by reverse transcription and real-time PCR (RT-PCR) of the corresponding cDNA and normalized to GAPDH, as described in the Materials and Methods section. Remarkably, imiquimod significantly reduced ( a ) adiponectin while increasing ( c ) MCP1/CCL2 mRNA levels in adipocytes in co-culture with monocytes; ( b ) Resistin mRNA levels were not significantly affected. (n.s.—not significant).

Article Snippet: After isolation of mRNA applying the RNeasy ® Mini Kit (Qiagen, Hilden, Germany), the gene expression levels of murine TLR7, TLR9, adiponectin, resistin, CD45, glucose transporter 1 (GLUT1) and glucose transporter 4 (GLUT4) and of human TLR7 and TLR9 were quantified by reverse transcription and real-time PCR (RT-PCR) of the corresponding cDNA.

Techniques: Co-Culture Assay, In Vitro, Isolation, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction