Journal: PLOS One

Article Title: Bacterial RNA downregulates MHC-I in tumor cell lines, promoting NK response and delaying tumor growth

doi: 10.1371/journal.pone.0357999

Figure Lengend Snippet: Different tumor cell lines were attached to a 48-well plate for 24 h. Then, cells were stimulated with ORN06 (10 μg/ml) for 48 h in the presence of IFN-γ. MHC-I expression in (A) U251, (B) LN-229, (C) HT-29, and (D) B16-OVA was determined by flow cytometry. Cells treated with IFN-γ were used as a positive control. Bars indicate geometric mean ± SEM of at least three experiments. MFI, mean fluorescence intensity; (-) untreated cells. * P < 0.05; *** P < 0.001 vs . IFN-γ-treated cells.

Article Snippet: For viability assays, U251, LN-229, HT-29, HCT, MCF-7, and B16-OVA cell lines were treated with bacRNA or the TLR8 ligand ORN06 (InvivoGen) in the presence of IFN-γ (7.5 ng/ml) for 48 h. Cells were washed with PBS and resuspended in diluted 25 μL Zombie Violet solution to check the viability of the treated cells.

Techniques: Expressing, Flow Cytometry, Positive Control, Fluorescence

Journal: PLOS One

Article Title: Bacterial RNA downregulates MHC-I in tumor cell lines, promoting NK response and delaying tumor growth

doi: 10.1371/journal.pone.0357999

Figure Lengend Snippet: (A) Experimental design to characterize immune responses following ORN06-based melanoma model treatment. Intratumoral ORN06 injections were administered after B16-OVA cell implantation (n = 4 per group). (B) Representative flow cytometry dot plot of the results shown in C-G. Detection of tumor infiltration and activation of dendritic cells (C), monocytes (D), NK cells (E), helper T cells (F) and CD8 T cells (G) by flow cytometry. Tumor cells were attached on a 96-well plate. After 24 h, supernatants were analyzed for the secretion of IFN-γ by sandwich ELISA (H). (E) Tumor growth curves of the intratumoral ORN06 treatment group vs . saline control group in the B16-OVA melanoma model, based on the experimental design in A. s.c.: subcutaneous injection; i.t.: intratumoral injection; ns, not statistically significant; * P < 0.05 vs . saline-treated tumor cells in D (panel right) and G (panel left); ** P < 0.01 vs . saline-treated tumor cells in E and F.

Article Snippet: For viability assays, U251, LN-229, HT-29, HCT, MCF-7, and B16-OVA cell lines were treated with bacRNA or the TLR8 ligand ORN06 (InvivoGen) in the presence of IFN-γ (7.5 ng/ml) for 48 h. Cells were washed with PBS and resuspended in diluted 25 μL Zombie Violet solution to check the viability of the treated cells.

Techniques: Flow Cytometry, Activation Assay, Sandwich ELISA, Saline, Control, Injection

Journal: bioRxiv

Article Title: Let-7b-5p differentially regulates human first trimester trophoblast migration and sFlt-1 through TLR7 and TLR8

doi: 10.64898/2026.08.03.742516

Figure Lengend Snippet: (A-C) Trophoblast cells were transfected with a scramble control (control), a Let-7b-5p mimic or a Let-7b-5p inhibitor. (A) After 24 hrs and 72 hrs, Let-7b-5p expression was measured by RT-qPCR and expressed as fold change (FC) relative to the control (n=6-9; * p <0.05). (C) After 48 hrs trophoblast cell viability was measured (n=4). (C) After 48 hrs trophoblast cell migration was measured (n=6-8; * p <0.05). (D-E) Trophoblast cells were treated: (D) for 24 hrs with no treatment (NT), the TLR7 inhibitor, IRS661, or the TLR8 inhibitor, CUCPT9a (n=9; * p <0.05), or (E) for 48hrs with NT, the TLR7 agonist, R837, or the TLR8 agonist TL8-506 (n=14; * p <0.05), after which cell migration was measured. (F) Trophoblast cells were transfected with a scramble control or a Let-7b-5p mimic and then treated with either media, IRS661 or CUCPT9a. After 24 hrs, cell migration was measured and data are shown as the fold change (FC) of trophoblast migration under Let-7b-5p mimic conditions relative to the scramble control (n=6; * p <0.05). (G) Trophoblast cells were treated with NT, IRS661 or CUCPT9a and after 24 hrs, cell viability was measured (n=3).

Article Snippet: In other experiments, trophoblast cells, in the absence of any transfection, were treated with either no treatment (NT), IRS661 at 5μM, CUCPT9a at 2.5 μM, the TLR7 agonist, R837 at 5μg/ml (Invivogen), or the TLR8 agonist, TL8-506 at 5μg/ml (Invivogen).

Techniques: Transfection, Control, Expressing, Quantitative RT-PCR, Migration

Journal: International Journal of Molecular Sciences

Article Title: Toll-like Receptor 8 (TLR8) Is Expressed in Mouse Hippocampal Neurons and Modulates Neuronal Excitability

doi: 10.3390/ijms27146232

Figure Lengend Snippet: Stimulation with the selective TLR8 agonist TL8-506 increases activity of mouse postnatal hippocampal cultures. ( A ) Scheme of the MEA recording protocol for TLR8 stimulation in hippocampal neuronal cultures. Baseline activity (BS) was recorded for 2 min, followed by cumulative stimulation with TL8-506 at concentrations. Neuronal cultures were then incubated with TL8-506 (1.0 µg/mL) at 37 °C with 5% CO 2 for 2 h. Baseline activity was again measured for 3 min after incubation with the agonist. To exclude electrodes that gave a false positive signal, the voltage-gated sodium channel blocker tetrodotoxin (TTX) (1 µM) was applied at the end of the recordings. ( B ) Representative traces of one HD-MEA recording, showing a single channel. ( C ) Chemical structure of the TLR8 selective agonist TL8-506 (Invivogen, San Diego, CA, USA) and analyzed parameters of MEA recording after TLR8 stimulation. Increased neuronal activity is reflected by augmented firing rate (spikes/s), bursting rate (bursts/min), and higher network synchrony (mean % of spikes in bursts) compared to unstimulated baseline activity of the neurons. Results are displayed as mean ± SEM, with the number of independent postnatal neuronal cultures ( N ) being 5, and the number of recorded channels for each recording being 452, 805, 1461, 1150, and 1093. The data were analyzed using one-way ANOVA, and normalization was performed by dividing the raw values through the mean baseline activity of the MEA chip. ( D ) Inhibition of TLR8 with CU-CPT9a decreased neuronal activity back to baseline levels. Results are displayed as mean ± SEM, with the number of independent postnatal neuronal cultures ( N ) being 2 and the number of recorded channels being 2864 and 2161. **** p ≤ 0.0001; ns, non-significant, p ≥ 0.05.

Article Snippet: CU-CPT9a , TLR8 antagonist , Invivogen , inh-cc9a , CU-CPT9a solvent.

Techniques: Activity Assay, Incubation, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Toll-like Receptor 8 (TLR8) Is Expressed in Mouse Hippocampal Neurons and Modulates Neuronal Excitability

doi: 10.3390/ijms27146232

Figure Lengend Snippet: Calcium imaging of primary hippocampal neurons following TL8-506 stimulation and TLR8 inhibition. ( A ) The activity of TL8-506 was inhibited by CU-CPT9a or the cells were treated with the specific vehicle used to dissolve CU-CPT9a. For comparison the activity obtained with ionomycin is indicated. N = 3. ( B ) Effect of D-AP5 on TL8-506-induced Ca 2+ -responses. The data was normalized within each plate to the respective ionomycin values. N = 3. Bars indicate mean ± SEM. Statistical analyses were performed using repeated measures one-way ANOVA with planned pairwise comparisons as indicated. ns, non-significant p ≥ 0.05; * p < 0.05; ** p < 0.01.

Article Snippet: CU-CPT9a , TLR8 antagonist , Invivogen , inh-cc9a , CU-CPT9a solvent.

Techniques: Imaging, Inhibition, Activity Assay, Comparison

Journal: Nature Communications

Article Title: A tissue-intrinsic mechanism sensitizes HIV-1 particles for TLR-triggered innate immune responses

doi: 10.1038/s41467-026-72586-3

Figure Lengend Snippet: a Schematic representation of PRRs and their cognate ligands. PRRs: green, adapter proteins: pink, PAMPs: red, transcription factors: blue. b PRR inhibitor screening. MDMs were challenged with S or DC HIV-1 NL4.3 R5 virions in presence or absence of PRR inhibitors. IL-6 release was measured by ELISA from MDM supernatants. Data is normalized to mock infected condition (gray dotted line). c Measurement of IL-6 release from the supernatants of MDMs challenged with S or DC HIV-1 virions in presence or absence of the indicated inhibitors. d Volcano plot illustrating the differential gene expression between MDMs challenged with DC virus and mock challenged cells for 6 h. The number of down- and upregulated genes are indicated. e Volcano plot as in d. with highlighted gene signatures. Genes induced by TLR2 alone (blue), TLR8 alone (brown), co-regulated (light green) or associated with “Innate Immune Response” genes (red). f Volcano plot illustrating the differential gene expression between MDMs challenged with suspension or collagen primed HIV-1 virus for 6 h. The number of down- and upregulated genes are indicated. g Volcano plot as in f. with highlighted gene signatures. Genes induced by TLR2 alone (blue), TLR8 alone (brown), co-regulated (light green) or associated with Innate Immune Response genes (red). Top deregulated genes belonging to different gene ontology terms are highlighted. h Gene ontology analysis of biological processes repressed or induced in MDMs challenged with collagen primed virions as compared to cells challenged with suspension virus for 6 h. FDR is indicated (only upregulated pathways have a q -value < 0.05). Top deregulated genes for each pathway are highlighted. Results represent the mean ± SD of 3 independent donors. Symbols indicate individual donors ( b , c ) or individual genes ( d–g ). Significance is indicated by p-values, and was calculated by two-tailed paired t-tests or Wilcoxon matched-pairs signed rank test ( b , c ), differential gene expression analysis was performed using DESeq2: genes with adjusted p-value < 0.05 were considered significant ( d–g ), for GSEA analysis, p -values were corrected using Benjamini-Hochberg FDR ( h ). Source data are provided as a file.

Article Snippet: 5 μM of cGAS inhibitor (G140, Invivogen), 8 μM of TLR 1/2 inhibitor (Cu-CPT22, Selleckchem), 49 μM of TLRs 4& 2/6 inhibitor (GIT27, Tocris), 2 μM of TLR4 inhibitor (CLI-095, Invivogen), 100 μM of TLR2 inhibitor (TL2-C29, Invivogen) or 10 μM of TLR8 inhibitor (Cu-CPT9a, Invivogen) were incubated with MDMs 3 h prior to infection.

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Gene Expression, Virus, Suspension, Two Tailed Test

Journal: Nature Communications

Article Title: A tissue-intrinsic mechanism sensitizes HIV-1 particles for TLR-triggered innate immune responses

doi: 10.1038/s41467-026-72586-3

Figure Lengend Snippet: a Representative structure of an HIV-1 Env trimer. Epitopes targeted by different bNAbs or nNAbs as well as sCD4 are highlighted (based upon a prediction of NL4.3 Env trimer structure using AlphaFold 3). b Experimental workflow. HIV-1 NL4.3 R5 Vpr.Int.GFP virions harvested from suspension or collagen cultures were sequentially incubated with primary, epitope-specific anti-gp120 antibodies, then with AF568 coupled secondary antibodies prior to processing for microscopy analysis. c . Representative micrographs of stained virions. Vpr.Int.GFP positive spots (green) can be seen bound by different antibodies or sCD4-PE (red). Scale bar: 0.5 µm. d Spider plot representing the binding frequency of antibody binding to HIV-1 NL4.3 R5 as in (c). e . sTLR2 is retained at the surface of NL4.3 R5 but not NL4.3 ΔEnv virions after collagen priming. One representative Western Blot analysis showing the ratio from HIV-1 NL4.3 virions cultured in S, DC or LC. The TLR2/p24 ratios are indicated below the blots after correction, after subtraction of the TLR2 background signals from the last two lanes. f Quantification of western blot analyses from three independent experiments as in e . g . Representative micrographs. MDMs challenged with HIV1 NL4.3 R5 Vpr.Int.GFP virions after culture in suspension or in DC. Yellow arrows indicate Vpr.Int.GFP/TLR8 colocalization. Scale bar: 5 µm. h Quantification of Virus/TLR8 colocalization from micrographs as in d. Results represent the mean ± SD of three independent experiments. Significance is indicated by p -values, and was calculated by two-tailed paired t-tests ( d , f , h ). Source data are provided as a file.

Article Snippet: 5 μM of cGAS inhibitor (G140, Invivogen), 8 μM of TLR 1/2 inhibitor (Cu-CPT22, Selleckchem), 49 μM of TLRs 4& 2/6 inhibitor (GIT27, Tocris), 2 μM of TLR4 inhibitor (CLI-095, Invivogen), 100 μM of TLR2 inhibitor (TL2-C29, Invivogen) or 10 μM of TLR8 inhibitor (Cu-CPT9a, Invivogen) were incubated with MDMs 3 h prior to infection.

Techniques: Suspension, Incubation, Microscopy, Staining, Binding Assay, Western Blot, Cell Culture, Virus, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Human neutrophils recognize group B streptococci via formylated peptide receptors and toll-like receptor 8

doi: 10.3389/fimmu.2026.1828994

Figure Lengend Snippet: TLR8 is required for GBS-induced cytokine production. Neutrophils were treated with the TLR8 inhibitor CU-CPT9a (0.75, 1.50 and 3.00μM) or the TLR2 inhibitor TL2-C29 (5, 10 and 25μM) before stimulation with live [MOI of 5, (A, B) ] or heat-killed GBS [25 μg/ml, (C, D) ]. IL-8 (A, C) and TNF-α (B, D) were measured in 24h culture supernatants. Escherichia coli lipopolysaccharide (LPS; 10 ng/mL) was included as a positive control. (E) Effect of pretreatment with the TLR8 CU-CPT9a inhibitor (3μM) on the release of reactive oxygen species after stimulation with live GBS (MOI 100). Data are expressed as means ± standard deviations from five independent experiments, each performed in duplicate. *p < 0.05 and **p < 0.01, as determined by the Mann-Whitney test; ns, not significant.

Article Snippet: The contribution of FPRs and TLRs to IL-8 and ROS production induced by live bacteria was evaluated by pretreating neutrophils for 1 hour at 37 °C in 5% CO 2 with the selective FPR2 antagonist WRW4 (Abcam), the pan-FPR inhibitor Boc-2 (GenScript), the selective TLR8 inhibitor CU-CPT9a (InvivoGen) or the selective TLR2 inhibitor TL2-C29 (InvivoGen) at the indicated concentrations prior to stimulation with heat-killed (HK) or live bacteria.

Techniques: Positive Control, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Human neutrophils recognize group B streptococci via formylated peptide receptors and toll-like receptor 8

doi: 10.3389/fimmu.2026.1828994

Figure Lengend Snippet: Cytokine responses to GBS by nucleic acids. IL-8 (A) and TNF-α (B) responses to stimulation with graded doses of GBS nucleic acids (0.01, 0.1, 1 and 10 µg/ml). In experiments involving pretreatment with the TLR8 inhibitor CU-CPT9a (3 μΜ), neutrophils were then stimulated with 10 µg/ml of nucleic acids (RNA or DNA). Data are expressed as means ± standard deviations from three independent experiments, each performed in duplicate. *p < 0.05, as determined by the Mann-Whitney test.

Article Snippet: The contribution of FPRs and TLRs to IL-8 and ROS production induced by live bacteria was evaluated by pretreating neutrophils for 1 hour at 37 °C in 5% CO 2 with the selective FPR2 antagonist WRW4 (Abcam), the pan-FPR inhibitor Boc-2 (GenScript), the selective TLR8 inhibitor CU-CPT9a (InvivoGen) or the selective TLR2 inhibitor TL2-C29 (InvivoGen) at the indicated concentrations prior to stimulation with heat-killed (HK) or live bacteria.

Techniques: MANN-WHITNEY