Fig 2: TLR2 and TLR3 play differential roles in EGCG-mediated ALD protection. (A) Serum chemistry for ALT and AST of Tlr2–/– and Tlr3–/– mice (n = 5). (B) Representative hepatic hematoxylin and eosin and Oil Red O staining of Tlr2–/– and Tlr3–/– mice. (C) Serum levels of cytokines measured by using enzyme-linked immunosorbent assay (n = 5). (D) Representative hepatic F4/80+ cell staining results of Tlr2–/– and Tlr3–/– mice. (E) Whole liver tissue immunoblotting results of Tlr2–/– and Tlr3–/– mice. (F) Real-time PCR results of key M1 and M2 markers from isolated Kupffer cells from Tlr2–/– and Tlr3–/– mice treated with ethanol diet or EGCG (n = 4). Scale bar = 20 μm. Values are expressed as mean ± SD (Three independently repeated experiments with similar results). *, ***P < .05 and .001 against pair group; #, ##, ###P < .01, .05, and .001 against EtOH (ALD) group. For panel F: *, **, ***P < .05, .01, and .001 between indicated groups. Statistical comparisons between groups were done by using the Kruskal-Wallis test followed by Dunn’s post hoc test to detect differences in all groups.

Fig 3: Increased TLR3 expression results in augmented MIP-2 expression in the lung.The results show the effects of sequential challenges with LPS and Poly I:C on MIP-2 expression in the lungs. In WT mice, LPS (5 μg/g) or saline (SAL) was injected intratracheally at time 0 h, and 4 hours later, Poly I:C (5 μg/g) or SAL was injected. In TLR4−/− and TLR3−/− mice, LPS was injected intratracheally (5 μg/g) at time 0 h, and Poly I:C (5 μg/g) was injected intratracheally at 4 h. Lung tissue was harvested at the indicated times, and MIP-2 protein expression was detected using Western blotting (A,C), with actin being used to normalize the densitometry of MIP-2 expression. Additionally, MIP-2 mRNA expression was detected using RT-PCR (B), and qRT-PCR was used to detect MIP-2 mRNA expression in AMΦ (D). β-actin mRNA was used to normalize the value of MIP-2 mRNA expression. The graphed values represent the mean ± SEM. Five mice were analyzed per group. **P < 0.01 compared with the other groups; ***P < 0.001 compared with the other groups.

Fig 4: Site-Directed Mutagenesis and Luciferase Analysis of TLR3 and IRF3 Mutations. (A and B) HEK293T cells were transiently transfected with IFNB1 firefly luciferase reporter, Renilla luciferase reporter, and plasmids encoding WT or Mutated (Arg488* TLR3, His684Arg TLR3, or Val363Met IRF3 as indicated). Cells were stimulated or not with 25 μg/ml poly(I:C), and luciferase activities were measured 6 h later. Firefly luciferase activity was normalized to Renilla luciferase activity and presented as percentage stimulation in cells expressing Arg488* TLR3, His684Arg TLR3, or Val363Met IRF3 relative to cells expressing WT TLR3 or IRF3. Data from 3 independent experiments are shown. One-way analysis of variance (ANOVA) and post hoc Tukey multiple comparison analyses were applied. For all analyses, P-values <0.05 were considered significant. (C) TOPO-TA cloning was performed to determine the positioning of the TLR3 variants, whether they were located on the same allele or different alleles (cis or trans). The cloning results indicated that both variants were on the same allele (Created with BioRender.com).

Fig 5: Activation of TLR3 signaling by Au-antimiR-155 NCs. (A) Western blotting analysis of TLR3, NF-αB (p65), and both full-length and cleaved caspase-8 expression in HepG2 cells treated with antimiR-155, gold precursor plus antimiR-155, or the respective controls (n=3). (B) Densitometric quantification of data shown in (A). (C) qRT-RCR analysis of TLR3, p65, and caspase-8 expression in HepG2 cells (n=3). (D) Quantification of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) secretion by ELISA. HepG2 cells were co-incubated with gold precursor and antimiR-155 for 24 h. **P< 0.01; *P < 0.05; ns, no significant difference.