Extended Data Fig. 5: Locked nucleic acid (LNA) bases mediate antagonism of single-stranded RNA ligands of TLR8.
From: LDL delivery of microbial small RNAs drives atherosclerosis through macrophage TLR8
a) HEK293T cells over-expressing human TLR8, UNC93B1 and CD14 were pre-treated with vehicle (DOTAP) or corresponding DNA/LNA oligonucleotides (2.5 μg/mL) for 30 min and then stimulated with TLR8 nucleoside analogue agonist CL075 or TLR8 ORN agonists ssRNA40 or ORN06 (2 μg/mL) for 24 h (n = 5 biological replicates). Two-way ANOVA with Dunnett’s multiple comparison test (statistical significance relative to untreated within each group; **p < 0.0001). b) THP-1 macrophages were pre-treated + /- nt-LNA (1 µg/mL) for 45 min and treated with LPS (500 ng/mL), Poly I:C (1 µg/mL), CL075 (2.5 μg/ml), or ssRNA40 at 1 µg/mL (1:1, nt-LNA:ssRNA40) or 0.2 µg/ml (5:1) for 24 h (n = 3 biological replicates). Relative mRNA expression of IL1B, IL6 and TNF were then assessed by qPCR. For each treatment, the relative fold change of each treatment in the presence of nt-LNA was expressed as a percentage of the relative fold change of each treatment without nt-LNA pre-treatment (% inhibition). Two-way ANOVA, Benjamini, Krieger and Yekutieli FDR (Q = 0.05), **q < 0.01. c-d) Primary human CD14 + PBMC differentiated with GM-CSF and IFNγ were pre-treated with 2.5 μg/mL nt-LNA or vehicle (DOTAP) for 30 minutes and then stimulated with or without ssRNA40 (0.5 μg/mL) for 24 h. c) mRNA expression was quantified by qPCR (n = 4 biological replicates) d) Cytokine (IL-6) secretion was quantified by ELISA (n = 4 biological replicates). One-way ANOVA with Dunnett’s multiple comparison test. **p < 0.01. ***p < 0.001, ****p < 0.0001 (e) mRNA expression (n = 4 biological replicates), (f) cytokine secretion (n = 3 biological replicates) and (g) immunoblotting (representative image of three independent experiments) of BMDMs following up to 24 h treatment with IFNγ (100 U/mL) +/- 0.5 mg/ml nLDL in the presence or absence of 2.5 μg/mL nt-LNA. Two-way ANOVA, Benjamini, Krieger and Yekutieli FDR (Q = 0.05), *q < 0.05, **q < 0.01. Data are mean ± SEM. Numerical source data, statistics, exact p values and q values are provided.
