tlr8 inhibitor  (InvivoGen)

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: Journal of extracellular vesicles

Article Title: Cancer-Specific RNA Modifications in Tumour-Derived Extracellular Vesicles Promote Tumour Growth.

doi: 10.1002/jev2.70083

Figure Lengend Snippet: FIGURE 4 m6A levels in EVs regulate inflammatory responses via TLR8 in macrophages. Differentiated THP-1 cells (dTHP-1) were pretreated with or without CU-CPT-9a and CU-CPT-4a. (A) Whole-cell lysates obtained from dTHP-1 cells were subjected to Western blot analysis using an anti-phospho p65 antibody, anti-p65 antibody and anti-β-actin antibody. Representative images from three independent experiments are shown. Conditioned medium from dTHP-1 cells treated with CCD-841-CoN or HT29 small EVs was used for ELISA. TNF-α (B, left and C, left), and IL-6 (B, right and C, right). Values are presented as the mean ± SEM for each group. One-way ANOVA post hoc Tukey’s test; *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant. dTHP- 1 cells were pre-transfected with or without TLR8 siRNA. (D) Whole-cell lysates obtained from dTHP-1 cells treated with CCD-841-CoN small EVs or HT29 small EVs were subjected to Western blot analysis using an anti-phospho p65 antibody, anti-p65 antibody and anti-β-actin antibody. Representative images from three independent experiments are shown. Conditioned medium was used for the ELISA. TNF-α (E, left) and IL-6 (E, right). Values are presented as the mean ± SEM for each group. One-way ANOVA post hoc Tukey’s test; **p < 0.01, ***p < 0.001, ****p < 0.0001. dTHP-1 cells were pre- transfected with or without TLR3 siRNA. (F) Whole-cell lysates obtained from dTHP-1 cells treated with CCD-841-CoN small EVs or HT29 small EVs were subjected to Western blot analysis using an anti-phospho p65 antibody, anti-p65 antibody and anti-β-actin antibody. Representative images from three independent experiments are shown. Conditioned medium was used for ELISA. TNF-α (G, left) and IL-6 (G, right). Values are presented as the mean ± SEM for each group. One-way ANOVA post hoc Tukey’s test; ***p < 0.001, ****p < 0.0001; ns, not significant. (H) HT29 cells were cultured in a conditioned medium from dTHP-1 cells transfected with or without TLR8 siRNA and CCD-841-CoN small EVs or HT29 small EVs. Values are presented as the mean ± SEM for each group. One-way ANOVA post hoc Tukey’s test; *p < 0.05, **p < 0.01. HT29-derived EVs-RNA were pretreated with or without rALKBH5 and introduced into dTHP-1 cells transfected with or without TLR8 siRNA. TNF-α (I) and IL-6 (J) concentrations were measured via ELISA using conditioned medium from dTHP-1 cells. Values are presented as the mean ± SEM for each group. One-way ANOVA post hoc Tukey’s test; *p < 0.05, ***p < 0.001, ****p < 0.0001, ns, not significant. See also Figure S7.

Article Snippet: TLR8 agonist (Motolimod), TLR3 agonist (Poly(I:C)), TLR8 inhibitor (CU-CPT9a) and TLR3 inhibitor (CU-CPT-4a) were purchased from MedChemExpress (Monmonth Junction, NJ, USA). dTHP-1 cells (1 × 105 cells) were seeded in 24-well plates and treated with inhibitors for 48 h simultaneously with EVs or agonists.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Cell Culture, Derivative Assay

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

Journal: Journal of Extracellular Vesicles

Article Title: Cancer‐Specific RNA Modifications in Tumour‐Derived Extracellular Vesicles Promote Tumour Growth

doi: 10.1002/jev2.70083

Figure Lengend Snippet: m6A levels in EVs regulate inflammatory responses via TLR8 in macrophages. Differentiated THP‐1 cells (dTHP‐1) were pretreated with or without CU‐CPT‐9a and CU‐CPT‐4a. (A) Whole‐cell lysates obtained from dTHP‐1 cells were subjected to Western blot analysis using an anti‐phospho p65 antibody, anti‐p65 antibody and anti‐β‐actin antibody. Representative images from three independent experiments are shown. Conditioned medium from dTHP‐1 cells treated with CCD‐841‐CoN or HT29 small EVs was used for ELISA. TNF‐α (B, left and C, left), and IL‐6 (B, right and C, right). Values are presented as the mean ± SEM for each group. One‐way ANOVA post hoc Tukey's test; * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant. dTHP‐1 cells were pre‐transfected with or without TLR8 siRNA. (D) Whole‐cell lysates obtained from dTHP‐1 cells treated with CCD‐841‐CoN small EVs or HT29 small EVs were subjected to Western blot analysis using an anti‐phospho p65 antibody, anti‐p65 antibody and anti‐β‐actin antibody. Representative images from three independent experiments are shown. Conditioned medium was used for the ELISA. TNF‐α (E, left) and IL‐6 (E, right). Values are presented as the mean ± SEM for each group. One‐way ANOVA post hoc Tukey's test; ** p < 0.01, *** p < 0.001, **** p < 0.0001. dTHP‐1 cells were pre‐transfected with or without TLR3 siRNA. (F) Whole‐cell lysates obtained from dTHP‐1 cells treated with CCD‐841‐CoN small EVs or HT29 small EVs were subjected to Western blot analysis using an anti‐phospho p65 antibody, anti‐p65 antibody and anti‐β‐actin antibody. Representative images from three independent experiments are shown. Conditioned medium was used for ELISA. TNF‐α (G, left) and IL‐6 (G, right). Values are presented as the mean ± SEM for each group. One‐way ANOVA post hoc Tukey's test; *** p < 0.001, **** p < 0.0001; ns, not significant. (H) HT29 cells were cultured in a conditioned medium from dTHP‐1 cells transfected with or without TLR8 siRNA and CCD‐841‐CoN small EVs or HT29 small EVs. Values are presented as the mean ± SEM for each group. One‐way ANOVA post hoc Tukey's test; * p < 0.05, ** p < 0.01. HT29‐derived EVs‐RNA were pretreated with or without rALKBH5 and introduced into dTHP‐1 cells transfected with or without TLR8 siRNA. TNF‐α (I) and IL‐6 (J) concentrations were measured via ELISA using conditioned medium from dTHP‐1 cells. Values are presented as the mean ± SEM for each group. One‐way ANOVA post hoc Tukey's test; * p < 0.05, *** p < 0.001, **** p < 0.0001, ns, not significant. See also Figure .

Article Snippet: TLR8 agonist (Motolimod), TLR3 agonist (Poly(I:C)), TLR8 inhibitor (CU‐CPT9a) and TLR3 inhibitor (CU‐CPT‐4a) were purchased from MedChemExpress (Monmonth Junction, NJ, USA). dTHP‐1 cells (1 × 10 5 cells) were seeded in 24‐well plates and treated with inhibitors for 48 h simultaneously with EVs or agonists.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Cell Culture, Derivative Assay

Journal: Journal of Extracellular Vesicles

Article Title: Cancer‐Specific RNA Modifications in Tumour‐Derived Extracellular Vesicles Promote Tumour Growth

doi: 10.1002/jev2.70083

Figure Lengend Snippet: 5′‐half‐GlyGCC in tumour EVs promotes tumour growth in vivo. Differentiated THP‐1 cells (dTHP‐1) were pre‐transfected with or without TLR8 siRNA and 5′‐half‐GlyGCC. Conditioned medium from dTHP‐1 cells was used for the ELISA. TNF‐α (A) and IL‐6 (B). Values are presented as the mean ± SEM for each group. One‐way ANOVA post hoc Tukey's test; ** p < 0.01, *** p < 0.001. (C) HT29 cells were cultured in a conditioned medium from dTHP‐1 cells transfected with or without TLR8 siRNA and 5′‐half‐GlyGCC. Values are presented as the mean ± SEM for each group. One‐way ANOVA post hoc Tukey's test; ** p < 0.01, *** p < 0.001. (D) Experimental scheme for the introduction of 5′‐half‐GlyGCC into colon26 small EVs and subsequent analysis. (E) Results of quantitative PCR of 5′‐half‐GlyGCC‐loaded colon26 small EVs (GlyGCC(+) EVs). Values are presented as the mean ± SEM for each group. Unpaired t ‐test; ** p < 0.01. ELISA was conducted using conditioned medium from RAW264.7 cells treated with GlyGCC(+) EVs. TNF‐α (F) and IL‐6 (G) concentration. Values are presented as the mean ± SEM for each group. Unpaired t ‐test; * p < 0.05, ** p < 0.01. Colon26 cells were injected into BALB/c mice. PBS, Control EVs or GlyGCC(+) EVs were injected i.p. Representative xenograft tumour images (H) are shown. White scale bar, 1 cm. Tumour size was measured and calculated daily (I). Values are presented as the mean ± SEM for each group. One‐way ANOVA post hoc Tukey's test; * p < 0.05, *** p < 0.001. Colon26 cells were injected into BALB/c mice. PBS, Control EVs or GlyGCC(+) EVs and clodronate liposomes were injected i.p. Representative xenograft tumour images (J) are shown. White scale bar, 1 cm. Tumour size was measured and calculated every day (K). Values are presented as the mean ± SEM for each group. Unpaired t ‐test; ns, not significant. (L) Immunohistochemical analysis of TNF‐α, IL‐6 and F4/80 expression in tumour tissues of mice inoculated with small EVs obtained from GlyGCC(+) EVs and clodronate liposomes. Scale bar: 100 µm. Representative images of six (PBS, Control EVs, GlyGCC(+) EVs, and Control EVs with clodronate liposomes) or five (GlyGCC(+) EVs with clodronate liposomes) independent experiments are shown. H‐scores of TNF‐α (M, left) and IL‐6 (M, right) expression in GlyGCC(+) EV injection experiments. Values are presented as the mean ± SEM for each group. One‐way ANOVA post hoc Tukey's test; * p < 0.05, ** p < 0.01, ns, not significant. H‐score of TNF‐α (N, left) and IL‐6 (N, right) expression in GlyGCC(+) EVs combined with clodronate liposome injection. Values are presented as the mean ± SEM for each group. Unpaired t ‐test; ns, not significant. (O) H‐score of F4/80 expression in GlyGCC(+) EVs, with or without clodronate liposome injection. Values are presented as the mean ± SEM for each group. Unpaired t ‐test; ns, not significant. See also Figures .

Article Snippet: TLR8 agonist (Motolimod), TLR3 agonist (Poly(I:C)), TLR8 inhibitor (CU‐CPT9a) and TLR3 inhibitor (CU‐CPT‐4a) were purchased from MedChemExpress (Monmonth Junction, NJ, USA). dTHP‐1 cells (1 × 10 5 cells) were seeded in 24‐well plates and treated with inhibitors for 48 h simultaneously with EVs or agonists.

Techniques: In Vivo, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Real-time Polymerase Chain Reaction, Concentration Assay, Injection, Control, Liposomes, Immunohistochemical staining, Expressing