tlr7 agonist  (InvivoGen)

Journal: Journal of Molecular Histology

Article Title: Interleukin-1 Receptor-Associated Kinase 3 Attenuates Chondrocyte Senescence and Osteoarthritis via Inhibition of the TLR7/9–NF-κB Axis

doi: 10.1007/s10735-026-10804-4

Figure Lengend Snippet: illustrates the role of IRAK3 in modulating TLR7/9 signaling and reducing inflammation in chondrocytes. AT791 is a TLR7 and TLR9 inhibitor. The treatment concentration is 3 µM and the treatment time is 48 h. The TLR9 agonist used is ODN 1826, with a concentration of 1 µg/ml and a treatment time of 48 h. The TLR7 agonist used is Resiquimod, with a concentration of 1 µM and a treatment time of 48 h. Both were purchased from MedChemExpress, HY-124,603, HY-146,245 and HY-13,740. Panels A and B show Western blot analysis of TLR7 and TLR9 expression following IRAK3 knockdown and overexpression, respectively. Panels C through E present ELISA results indicating levels of IL-1β, IL-6, and TNF-α after IRAK3 knockdown. Similarly, panels F through H display ELISA findings for IL-1β, IL-6, and TNF-α levels following IRAK3 overexpression. In panel I, chondrocytes were treated with TLR7/9 inhibitors and agonists, and β-galactosidase staining was performed on senescent chondrocytes for statistical analysis. (* p < 0.05, ** p < 0.01)

Article Snippet: The NF-κB inhibitor BAY 11-7082, along with TLR7/9 agonists and inhibitors, were procured from MedChemExpress.

Techniques: Concentration Assay, Western Blot, Expressing, Knockdown, Over Expression, Enzyme-linked Immunosorbent Assay, Staining

Journal: bioRxiv

Article Title: Liver sinusoidal endothelial cells integrate metabolic and immune signals for MAPK-dependent BMP6 regulation and hepcidin induction

doi: 10.64898/2026.05.07.723498

Figure Lengend Snippet: (A) Bmp6 mRNA expression in primary LSECs treated with 5 ng/mL LPS or 2.5 μM heme for 6 h in the presence or absence of hepatocyte-conditioned medium. (B) Bmp6 mRNA expression in primary LSECs treated with various TLR ligands: Pam3CSK4 (TLR1/2), PGN (TLR2), Poly I:C (TLR3), LPS (TLR4), FLA-ST (TLR5), FSL1 (TLR2/6), R848 (TLR7/8), and ODN (TLR9) or vehicle control (NT, non-treated) for 6 h. (C-D) Bmp6 mRNA expression in primary LSECs treated with increasing concentrations of LPS for 6 h or with 5 ng/mL LPS for 2, 4, and 6 h. (E) Bmp6 mRNA expression in LSECs treated with 2.5 μM heme or protoporphyrin IX (PPIX) for 6 h. (F-G) Bmp6 mRNA expression in primary LSECs treated with increasing concentrations of heme for 6 h or treated with 2.5 μM heme for 2, 4, or 6 h. (H) Bmp6 mRNA expression in primary LSECs pre-treated with TAK242 (5 μM) or DMSO for 1 h, followed by 5 ng/mL LPS or 2.5 μM heme treatment for 6 h. (I) Transcription factor activity analysis by RNA-seq of LSECs treated with heme with respect to untreated controls (contrast-wise), in presence of hepatocyte-conditioned medium. (J) Bmp6 mRNA expression in primary LSECs pre-treated with CHX (5 μM) or DMSO for 1 h, followed by 5 ng/mL LPS treatment for 6 h. Cell culture experiments, except those in panel A, were always conducted in the presence of hepatocyte-conditioned medium. Gene expression levels were assessed by RT-qPCR, normalized to the housekeeping gene Rpl19 , and expressed as fold change relative to vehicle-treated controls. The dashed line (ut) represents the mRNA expression of LSECs treated with the conditions shown, in the absence of LPS or heme. Data are obtained from three or four independent experiments and displayed as mean ± SD. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA or Student’s t-test. CM, conditioned medium; PPIX, protoporphyrin IX; CHX, cycloheximide.

Article Snippet: The TLR ligands Pam3CSK4 (TLR2:1) (#tlrl-pms), PGN-SA (TLR2) (#tlrl-pgns2), Poly I:C (TLR3) (#tlrl-picw), FLA-ST (TLR5) (#tlrl-stfla), FSL1 (TLR2:TLR6) (tlrl-fsl), R848 (TLR7:8) (#tlrl-r848-1), ODN (TLR9) (#tlrl-1826) and the MAPK inhibitor SP600125 (#tlrl-sp60) were purchased form Invivogen and diluted in PBS (TLR ligands) or DMSO (SP600125).

Techniques: Expressing, Control, Activity Assay, RNA Sequencing, Cell Culture, Gene Expression, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: De novo COVID-19-associated insulin resistance drives dysregulated neutrophil extracellular trap formation (NETosis) four months after infection

doi: 10.3389/fimmu.2026.1787799

Figure Lengend Snippet: Evaluation of NETosis in neutrophils of the study cohort by flow cytometry. (A) Flow cytometry plot strategy for the identification of PMN populations, neutrophils (using CD-16/anti-Singlec8 staining), quantification of TLR7/8 and identification of NETosis (using Live/Dead Dye and Sytox Blue staining). (B) Scatter plots showing the quantification of the percentage of baseline NETosis in neutrophils from the three patient groups of the study cohort. Ordinary one-way ANOVA *p <0.05. (C) Paired data scatter plots comparing the percentage of baseline NETosis and when stimulated with IL-6 (20ng/mL) and TNFα (2ng/mL). 2-way ANOVA **p <0.01. Scatter plots showing the quantification of the expression of (D) TLR7 and (E) TLR8 according to median fluorescence intensity (MFI). Ordinary one-way ANOVA. (F) Scatter plots of paired data comparing the percentage of baseline NETosis against stimuli on TLR receptors such as ImiQ (2μg/mL), (G) R848 (2μg/mL), (H) CL075 (1μg/mL), (I) ssRNA40 (1μg/mL) and (J) ssRNA41 (1μg/mL). For panels (F–J) , 2-way ANOVA *p <0.05, **p <0.01 ***p <0.005.

Article Snippet: For NETosis, 1 x 10 6 cells were incubated for 30 minutes at 37 °C and 5% CO2 under basal conditions or using IL-6 (20ng/mL) and TNFα (2ng/mL), or TLR7/8 agonists (CL075 (1μg/mL), ImiQ (2μg/mL), and R848 (2μg/mL), ssRNA40 (1μg/mL) and ssRNA 41(1μg/mL) (Catalog No.tlrl-kit3hw3, InvivoGen).

Techniques: Flow Cytometry, Staining, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Defining the Immunomodulatory Determinants of 3-mer Oligonucleotides on TLR7 and TLR8 sensing

doi: 10.64898/2026.04.29.720527

Figure Lengend Snippet: ( a , e and h ) HEK-TLR7 and HEK-TLR8 cells were pre-treated ∼30 min with 125 nM (TLR7) and 5 μM (TLR8) of the indicated oligos prior to overnight stimulation with 1 μg/ml of R848 followed by luciferase assay. Data are mean of n=3 independent experiments. Data were background-corrected using the non-treated (NT) condition and are shown as relative expression to R848 only (± s.e.m. and one-way ANOVA with uncorrected Fisher’s LSD tests shown compared to GUC-v1+R848 [ a (TLR7)], GUC+R848 [ a (TLR8), and e ] and R848 [ h ] condition; a [TLR7 and TLR8]: P<0.0001 ; e [TLR7]: P<0.0001 and [TLR8]: P=0.0005 ; h [TLR7 and TLR8]: P<0.0001 ). ( c , g and j ) HEK-TLR7 cells were pre-treated ∼30 min with 50 nM of the indicated oligos prior to overnight stimulation with 1 μg/ml of R848 followed by luciferase assay. Data are mean of n=3 independent experiments. Data were background-corrected using the non-treated (NT) condition and are shown as relative expression to R848 only (± s.e.m. and one-way ANOVA with uncorrected Fisher’s LSD tests shown compared to GUC+R848 ( c and g ) and GUC-v1+R848 ( j ) condition; c , g and j : P<0.0001 ). ( d ) HEK-TLR7 cells were pre-treated ∼30 min with different doses (1000 nM, 500 nM, 250 nM, 125 nM and 62.5 nM) of the indicated oligos and Enpatoran (100 nM, 50 nM, 25 nM, 12.5 nM and 6.25 nM) prior to overnight stimulation with 1 μg/ml of R848 followed by luciferase assay. Data are mean of n=3 independent experiments. Data were background-corrected using the non-treated (NT) condition and are shown as relative expression to R848 only (± s.e.m. and one-way ANOVA with uncorrected Fisher’s LSD tests shown compared to R848 only condition).

Article Snippet: HEK-BlueTM TLR7 (Invivogen, hkb-htlr7v2) reporter cells were cultured according to the manufacturer’s instructions and plated in complete DMEM prior to treatment.

Techniques: Luciferase, Expressing

Journal: bioRxiv

Article Title: Defining the Immunomodulatory Determinants of 3-mer Oligonucleotides on TLR7 and TLR8 sensing

doi: 10.64898/2026.04.29.720527

Figure Lengend Snippet: ( a-c ) Surface plasmon resonance (SPR) analyses of recombinant wild type human TLR8 ( a , b ), wild type mmTLR7 ( c ), mutant TLR8 (F495S) or mutant mmTLR7 (F507S) with the indicated concentrations of indicated 3-mers. Data shown are representative of 3-4 independent analyses (Supplementary Table S3). ( d, e ) Cryo-EM structure of the TLR7/GUC-v16 complex and crystal structure of the TLR8/GUC-v16 complex. Two TLR7 or TLR8 protomers and GUC-v16 are shown in cartoon and sphere representations, respectively. ( f, g ) Close-up views of the GUC-v16 (mImUmC PS ) recognition at the antagonistic sites of TLR7 and TLR8. Residues within 4.5 Å from GUC-v16 are shown in stick representations. Sticks are colored by atoms, with the N, O, P and S atoms colored by blue, red, orange and yellow, respectively. Yellow dashed lines indicate hydrogen bonds (cutoff distance < 3.5 Å). ( h ) Structural alignment of TLR7/GUC-v16 and TLR8/GUC-v16 complexes. Structural alignment was performed using the Matchmaker tool in ChimeraX.

Article Snippet: HEK-BlueTM TLR7 (Invivogen, hkb-htlr7v2) reporter cells were cultured according to the manufacturer’s instructions and plated in complete DMEM prior to treatment.

Techniques: SPR Assay, Recombinant, Mutagenesis, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Defining the Immunomodulatory Determinants of 3-mer Oligonucleotides on TLR7 and TLR8 sensing

doi: 10.64898/2026.04.29.720527

Figure Lengend Snippet: ( a ) Bone-marrow-derived macrophages (BMDMs) from Tlr7 Y264H mice were treated for 24 h with 1 μM of BMS905 and 5 μM of GUC-v16 oligo prior to RNA purification for RNA-sequencing or RT-qPCR analyses. RT-qPCR analyses of Fpr1, Slc13a3, Cd300e, Itgal, Nfkbiz, Slamf9, Clec4a2 and Clec4a1 reported to 18S in RNA lysates from primary BMDMs from 3 independent Tlr7 Y264H mice. Data are shown relative to non-treated (NT) mice (± s.e.m. and two-way ANOVA with uncorrected Fisher’s LSD tests shown compared to NT Tlr7 Y264H condition). ( b ) WT C57/BL6 mice were injected i.v. with 200 μg of GUC-v16 conjugated with in vivo -jetPEI® or vehicle (glucose solution) for 1 h prior to i.p. injection of 25 μg R848 for 2 h before collection of spleens. RT-qPCR analyses of Fpr1, Fpr2, Marco, Nfkbiz and Tnf reported to Gapdh , from spleen lysates; data are shown relative to non-treated (NT) mice (± s.e.m. and two-way ANOVA with uncorrected Fisher’s LSD tests shown compared to R848 mice). Each dot represents an individual mouse with bars showing the mean of n=5 mice/group is shown (± s.e.m. and one-way ANOVA with uncorrected Fisher’s LSD tests shown compared to R848 group). ( c ) Aldara cream was applied topically to the back of WT C57/BL6 mice directly following, or not, application of 100 μl of highly pure 2.5% GUC-v16 cream oligonucleotide (>99.4%). After four days, mice were humanely euthanised and back skin collected, and lysed for RNA purification. RT-qPCR analyses of indicated genes reported to that of 18S expression, relative to NT mice. Data are representative of 2 independent experiments. Mean of n=3 NT and n=8 Aldara/Aldara+GUC-v16 mice/group is shown (± s.e.m. and one-way ANOVA with uncorrected Fisher’s LSD tests shown compared to Aldara group). ( d-f ) Wild type C57BL/6J mice were treated i.t. with 2.5 μg of naked GUC-v16 in water for 1 h prior to. i.t. injection of 50 μg of R848. BALF ( d, f ) were harvested 7 h post R848 injection and analysed by cytospin differential cell counting for total cell counts ( d ) and using an MSD multiplex assay for cytokine quantification ( f ). Cytokine levels in plasma were also quantified using an MSD multiplex assay ( e ). Data shown are from 1 experiment, representative of n=2 independent experiments. Mean of n=5 NT and n=7 R848/R848+GUC-v16 mice/group is shown (± s.e.m. and one-way ANOVA with uncorrected Fisher’s LSD tests shown compared to NT [d] or R848 [ e,f ] group). Whole blood from 4 ( g ) or 1 ( h ) healthy controls or patients with UNC93B1 E92G mutation ( h ) was pre-treated with indicated 3-mer concentration for 30 min prior to stimulation with indicated amount of R837 or 50ng/ml TL8-506 for 24 h prior to cytokine bead analyses. ( g ) For each control, cytokine levels were averaged for each technical replicate (6/sample), background corrected to unstimulated samples only, and reported to cytokine levels from stimulation only controls. Heat maps were generated using scale from 0-1 (see methods), were 1 is the cytokine level of the agonist only control. ( g-h ) Data shown are from a minimum of two independent experiments – conducted on two independent days. ( i ) HEK-TLR7 cells were pre-treated with indicated concentration of oligo for 1 h prior to overnight stimulation with 10% of synovial fluid from RA patient. SEAP absorbances were background-corrected using the non-treated condition, and are shown as relative expression to the synovial fluid only control; Neg. is a non-oligo condition used as control. Data shown are averaged from synovial fluid stimulations from n=8 patients (± s.e.m. and two-way ANOVA with uncorrected Fisher’s LSD tests shown compared to Neg condition).

Article Snippet: HEK-BlueTM TLR7 (Invivogen, hkb-htlr7v2) reporter cells were cultured according to the manufacturer’s instructions and plated in complete DMEM prior to treatment.

Techniques: Derivative Assay, Purification, RNA Sequencing, Quantitative RT-PCR, Injection, In Vivo, Cream, Expressing, Cell Counting, Multiplex Assay, Clinical Proteomics, Mutagenesis, Concentration Assay, Control, Generated

Journal: Frontiers in Immunology

Article Title: Enhanced TLR7-induced interferon responses in women living with HIV

doi: 10.3389/fimmu.2026.1791579

Figure Lengend Snippet: PBMCs Of WLWH produce less inflammatory cytokines compared to PBMCs of MLWH Upon TLR7 activation. (a) Whole blood of 192 women living with HIV and 1134 men living with HIV was used to measure circulating immune cell distribution. Counts of monocytes and lymphocytes as well as the lymphocyte/monocyte Ratio between women and men living with HIV. Significance was tested using a Wilcoxon rank-sum test (b) PCA plot of cytokine production values in women and men. (c) PBMCs were isolated and stimulated with IMQ for 24 hours. Cytokines between women and men living with HIV were measured in the supernatant. Significance was tested using a logistic regression model, adjusted for age, season of inclusion, inclusion before or after COVID-19 pandemic, ancestry, and lymphocyte/monocyte ratio. P-values adjusted for multiple testing. Asterisks indicate statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: PBMCs were seeded at 500,000 cells/well in U-bottom plates (Corning, Corning, NY, USA) and subsequently stimulated with 5 μg/mL of the TLR7 agonist imiquimod (IMQ; Invivogen, San Diego, CA, USA) for 24 hours at 37 °C and 5% CO2.

Techniques: Activation Assay, Isolation

Journal: Frontiers in Immunology

Article Title: Enhanced TLR7-induced interferon responses in women living with HIV

doi: 10.3389/fimmu.2026.1791579

Figure Lengend Snippet: Baseline single-cell transcriptomics reveals sex-biased IRF7 expression in pDCs Of PLWH. (a) Dot plot depicting the relative expression and proportion of cells expressing selected genes related to TLR7 signaling, type I IFN responses, and inflammation across immune cell types. (b) Principal component analysis (PCA) of pseudobulked gene expression profiles from pDCs of women (n = 11) and men (n = 52) living with HIV. Percent variance explained by PC1 and PC2 is indicated. (c) Box plots showing batch-corrected normalized expression of TLR7 and IRF7 in pDCs, comparing women and men living with HIV. Pseudobulked data of pDCs was adjusted for Age, center of inclusion, inclusion before or after the COVID-19 pandemic, and vaccination against SARS-CoV-2. Significance was tested using a Wilcoxon rank-sum test. Asterisks indicate statistical significance: *P < 0.05.

Article Snippet: PBMCs were seeded at 500,000 cells/well in U-bottom plates (Corning, Corning, NY, USA) and subsequently stimulated with 5 μg/mL of the TLR7 agonist imiquimod (IMQ; Invivogen, San Diego, CA, USA) for 24 hours at 37 °C and 5% CO2.

Techniques: Single-cell Transcriptomics, Expressing, Gene Expression

Journal: Frontiers in Immunology

Article Title: Enhanced TLR7-induced interferon responses in women living with HIV

doi: 10.3389/fimmu.2026.1791579

Figure Lengend Snippet: Differential gene expression and pathway enrichment in TLR7−stimulated PBMCs highlight augmented type I/II interferon signatures in WLWH. (a) PBMCs of 27 women and 43 men living with HIV were stimulated with IMQ for 24 hours and then processed for bulk-RNA sequencing. Differential gene expression analysis revealed 43 up- and 40 downregulated genes in WLWH compared to MLWH upon IMQ stimulation. (b) Principal component analysis of top 5000 genes between WLWH and MLWH. (c) Differentially expressed genes in WLWH compared to MLWH upon IMQ stimulation based on FDR corrected p-values >0.05 and log2FoldChange. Genes from the X- and Y-chromosomes were excluded in this visualisation but can be found in the supplementary information. (d) Pathway enrichment analysis based on differentially expressed genes in WWH compared to MWH upon IMQ stimulation using the HALLMARK gene set.

Article Snippet: PBMCs were seeded at 500,000 cells/well in U-bottom plates (Corning, Corning, NY, USA) and subsequently stimulated with 5 μg/mL of the TLR7 agonist imiquimod (IMQ; Invivogen, San Diego, CA, USA) for 24 hours at 37 °C and 5% CO2.

Techniques: Gene Expression, RNA Sequencing

Journal: Frontiers in Immunology

Article Title: Enhanced TLR7-induced interferon responses in women living with HIV

doi: 10.3389/fimmu.2026.1791579

Figure Lengend Snippet: Impact of menopause status on cytokine production upon TLR7 stimulation in WLWH. (a) Number of WLWH categorised as pre-, peri- or post-menopausal according to self-reported questionnaire. (b) Plasma protein measurement of FSHB stratified for menopause status. (c) Cytokine production of PBMCs upon stimulation with IMQ stratified for menopause status. PBMCs were isolated and stimulated with IMQ for 24 hours. Cytokines between women living with HIV in different menopause stages were measured in the supernatant. Significance was tested using a logistic regression model, adjusted for age, season of inclusion, inclusion before or after COVID-19 pandemic, ancestry, and lymphocyte/monocyte ratio. P-values adjusted for multiple testing. Asterisks indicate statistical significance: ***P < 0.001, ****P < 0.0001.

Article Snippet: PBMCs were seeded at 500,000 cells/well in U-bottom plates (Corning, Corning, NY, USA) and subsequently stimulated with 5 μg/mL of the TLR7 agonist imiquimod (IMQ; Invivogen, San Diego, CA, USA) for 24 hours at 37 °C and 5% CO2.

Techniques: Clinical Proteomics, Isolation

Journal: Frontiers in Immunology

Article Title: Enhanced TLR7-induced interferon responses in women living with HIV

doi: 10.3389/fimmu.2026.1791579

Figure Lengend Snippet: Differential Gene Expression of PBMCs of HIC vs. non-HIC upon TLR7 Stimulation. (a) PCA analysis of gene expression levels based on HIV controller status and (b) sex. (c) PBMCs of 28 HIC and 35 non-HIC were stimulated with IMQ for 24 hours and then processed for bulk-RNA sequencing. Differential gene expression analysis revealed that no genes were up- or downregulated upon IMQ stimulation in HIC compared to non-HIC.

Article Snippet: PBMCs were seeded at 500,000 cells/well in U-bottom plates (Corning, Corning, NY, USA) and subsequently stimulated with 5 μg/mL of the TLR7 agonist imiquimod (IMQ; Invivogen, San Diego, CA, USA) for 24 hours at 37 °C and 5% CO2.

Techniques: Gene Expression, RNA Sequencing

Journal: iScience

Article Title: Comprehensive analysis of neutrophil immunomodulatory properties and FcR dynamics in HIV-1 infection and therapy

doi: 10.1016/j.isci.2026.115431

Figure Lengend Snippet: Activation of human neutrophils stimulated in vitro by Toll-like receptor (TLR) agonists Neutrophils from HD were activated overnight (20h) by TLR4 (LPS) or TLR7/8 (R848) agonists. Activated neutrophils and corresponding cell culture supernatants were collected to evaluate neutrophil viability and phenotypic activation via flow cytometry, as well as to analyze their secretome using bead-based immunoassays. (A) Gating strategy used for all experiments. (B and C) Viability (B) and phenotypic activation (C) by monitoring cell surface markers using flow cytometry. Boxplots (median, first and third quartiles, minimum and maximum) are used to represent selected parameters from 14 independent experiments. (D) Cytokines and chemokines secretion profile. Secretion of soluble cytokines (IL-1β, Il-6, TNF-α, CXCL10, IFN-λ1, IL-8, IL-12p70, IFN-α2, IFN-λ2/3, GM-CSF, IFN-β, IL-10, and IFN-γ) and chemokines (IL-8, CXCL10, CCL11, CCL17, CCL2, CCL5, CCL3, CXCL9, CXCL5, CCL20, CXCL1, CXCL11, and CCL4) was assayed from cell-free cell culture supernatants. Only cytokines/chemokines detected in cell culture supernatants are represented. Values are median with an interquartile range (IQR) of 8 independent measurements. Significance was assigned as follows: ∗ p < 0.05, ∗∗ p < 0,01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 NS: non-stimulated. (B and C) One-way ANOVA with Multiple comparisons (Friedman test). (D) One-way ANOVA with multiple comparisons (Dunn test).

Article Snippet: TLR7/8 agonist: R848 , Invivogen , cat #tlrl-r848.

Techniques: Activation Assay, In Vitro, Cell Culture, Flow Cytometry

Journal: iScience

Article Title: Comprehensive analysis of neutrophil immunomodulatory properties and FcR dynamics in HIV-1 infection and therapy

doi: 10.1016/j.isci.2026.115431

Figure Lengend Snippet: Activation of neutrophils isolated from HD or PLWH stimulated in vitro by TLR agonists LPS (TLR4) and R848 (TLR7/8) or inflammatory cytokines (TNF-α and IFN-γ) Neutrophils from HD were activated overnight (20h) by TLR agonists or pro-inflammatory cytokines. Cell culture supernatants of activated neutrophils were collected to analyze their secretome using bead-based immunoassays, as shown in . (A and B) Cytokines and chemokines secretion profile upon activation by TLR agonists, represented as a heatmap (A) and histograms (B) for each cytokine/chemokine assessed. Values are median with an IQR of at least 9 independent measurements. The heatmap in (A) represents data from all samples shown in (B). (C and D) Cytokine and chemokine secretion profile upon activation by inflammatory cytokines, represented as a heatmap (C) and histograms (D) for each cytokine/chemokine assessed. Values are median with an IQR of at least 9 independent measurements. The heatmap in (C) represents data from all samples shown in (D). Several cytokines and chemokines were only detected in a fraction of samples assayed. Significance was assigned as follows: ∗ p < 0.05 and ∗∗ p < 0.01. Kruskal-Wallis test for unpaired data with additional multiple comparisons, Dunn test.

Article Snippet: TLR7/8 agonist: R848 , Invivogen , cat #tlrl-r848.

Techniques: Activation Assay, Isolation, In Vitro, Cell Culture

Journal: iScience

Article Title: Comprehensive analysis of neutrophil immunomodulatory properties and FcR dynamics in HIV-1 infection and therapy

doi: 10.1016/j.isci.2026.115431

Figure Lengend Snippet: Activation of human neutrophils stimulated in vitro by Toll-like receptor (TLR) agonists Neutrophils from HD were activated overnight (20h) by TLR4 (LPS) or TLR7/8 (R848) agonists. Activated neutrophils and corresponding cell culture supernatants were collected to evaluate neutrophil viability and phenotypic activation via flow cytometry, as well as to analyze their secretome using bead-based immunoassays. (A) Gating strategy used for all experiments. (B and C) Viability (B) and phenotypic activation (C) by monitoring cell surface markers using flow cytometry. Boxplots (median, first and third quartiles, minimum and maximum) are used to represent selected parameters from 14 independent experiments. (D) Cytokines and chemokines secretion profile. Secretion of soluble cytokines (IL-1β, Il-6, TNF-α, CXCL10, IFN-λ1, IL-8, IL-12p70, IFN-α2, IFN-λ2/3, GM-CSF, IFN-β, IL-10, and IFN-γ) and chemokines (IL-8, CXCL10, CCL11, CCL17, CCL2, CCL5, CCL3, CXCL9, CXCL5, CCL20, CXCL1, CXCL11, and CCL4) was assayed from cell-free cell culture supernatants. Only cytokines/chemokines detected in cell culture supernatants are represented. Values are median with an interquartile range (IQR) of 8 independent measurements. Significance was assigned as follows: ∗ p < 0.05, ∗∗ p < 0,01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 NS: non-stimulated. (B and C) One-way ANOVA with Multiple comparisons (Friedman test). (D) One-way ANOVA with multiple comparisons (Dunn test).

Article Snippet: Cells were stimulated for 20h using TLR agonist, inflammatory cytokines or viral determinants under the following conditions: (i) TLR4 agonist (LPS ; Sigma-Aldrich, cat #L4524) or TLR7/8 agonist (R848 ; InvivoGen, cat #tlrl-r848) at 0,5 μg/mL each; (ii) IFN-γ (R&D, cat #285-IF-100) at 50ng/mL or TNF-α (Peprotech, cat #AF-300-01A) at 1ng/mL; (iii) free HIV-1 virions (Ad8 strain, 10 ng/mL of HIV-1 p24) or ICs made with HIV-1 Ad8 and the 10-1074 bNAb (as described below) or the 10-1074 bNAb alone (at 1μg/mL).

Techniques: Activation Assay, In Vitro, Cell Culture, Flow Cytometry

Journal: iScience

Article Title: Comprehensive analysis of neutrophil immunomodulatory properties and FcR dynamics in HIV-1 infection and therapy

doi: 10.1016/j.isci.2026.115431

Figure Lengend Snippet: Activation of neutrophils isolated from HD or PLWH stimulated in vitro by TLR agonists LPS (TLR4) and R848 (TLR7/8) or inflammatory cytokines (TNF-α and IFN-γ) Neutrophils from HD were activated overnight (20h) by TLR agonists or pro-inflammatory cytokines. Cell culture supernatants of activated neutrophils were collected to analyze their secretome using bead-based immunoassays, as shown in . (A and B) Cytokines and chemokines secretion profile upon activation by TLR agonists, represented as a heatmap (A) and histograms (B) for each cytokine/chemokine assessed. Values are median with an IQR of at least 9 independent measurements. The heatmap in (A) represents data from all samples shown in (B). (C and D) Cytokine and chemokine secretion profile upon activation by inflammatory cytokines, represented as a heatmap (C) and histograms (D) for each cytokine/chemokine assessed. Values are median with an IQR of at least 9 independent measurements. The heatmap in (C) represents data from all samples shown in (D). Several cytokines and chemokines were only detected in a fraction of samples assayed. Significance was assigned as follows: ∗ p < 0.05 and ∗∗ p < 0.01. Kruskal-Wallis test for unpaired data with additional multiple comparisons, Dunn test.

Article Snippet: Cells were stimulated for 20h using TLR agonist, inflammatory cytokines or viral determinants under the following conditions: (i) TLR4 agonist (LPS ; Sigma-Aldrich, cat #L4524) or TLR7/8 agonist (R848 ; InvivoGen, cat #tlrl-r848) at 0,5 μg/mL each; (ii) IFN-γ (R&D, cat #285-IF-100) at 50ng/mL or TNF-α (Peprotech, cat #AF-300-01A) at 1ng/mL; (iii) free HIV-1 virions (Ad8 strain, 10 ng/mL of HIV-1 p24) or ICs made with HIV-1 Ad8 and the 10-1074 bNAb (as described below) or the 10-1074 bNAb alone (at 1μg/mL).

Techniques: Activation Assay, Isolation, In Vitro, Cell Culture