Fig 2: MiRNAs miR-672-5p, let-7 g-5p, miR-100-5p, and miR-298-5p induce cytokine and chemokine release from microglia. Microglia isolated from C57BL/6 mice were incubated with 5 µg/ml of candidate miRNA, as indicated, for either 24 h (Gro-a, IFN-?, IL-6, MIP-2) or 36 h (other cytokines/chemokines). The TLR7 agonist loxoribine (1 mM) and the TLR4 agonist LPS (100 ng/ml) served as positive control. Unstimulated cells were used as negative control. The release of depicted inflammatory mediators was subsequently analyzed by multiplex immune assay. Data are shown as fluorescent intensity (FI) values expressed as a. u., arbitrary units, mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 compared to negative control, one-way ANOVA with Holm-Sidak’s multiple comparison test

Fig 3: ADEV miR-138 interacts with murine TLR7 in the endosomes. (A) Representative fluorescence images of mouse primary microglial cells incubated with Exo-Fect+ADEVs+Cy5-miR138 or Exo-Fect+Cy5-miR138 (without ADEVs) or Exo-Fect+ADEVs+miR138 (unstained) for 30 min followed by immunostaining of (A) an early endosome marker (EEA1, Green) and (B) TLR7 (Green). Exo-fect+ADEVs+Cy5-miR138, Cy5-miR138 were loaded into ADEVs using by Exo-Fect transfection kit; Exo-fect+Cy5-miR138, Cy5-miR138 were loaded using by Exo-Fect transfection kit (without ADEVs); Exo-fect+ADEVs+miR138, unstained miR138 were loaded into ADEVs using Exo-Fect transfection kit; Bars, 50 µm (n = 3). (C) TLR7 was immunoprecipitated from BV2 cells by IgG / TLR7 / LAMP1 antibody, followed by assessment of miR138 / U6 expression by real-time PCR. One-way ANOVA followed by Bonferroni's post hoc test was used to determine the statistical significance among multiple groups (n = 3). (D) TLR7 was immunoprecipitated from HEK-Null / HEK-TLR7 cells by IgG/TLR7 antibody, followed by assessment of miR138 expression by real-time PCR. One-way ANOVA followed by Bonferroni's post hoc test was used to determine the statistical significance among multiple groups (n = 3). (E) The protein of TLR7 were pull down by miR-138-biotin / miR-mut-138-biotin with Streptavidin agarose beads in BV2 cells. All data are presented as mean ± SD or SEM of three individual experiments. *,P < 0.05; **,P < 0.01; ***,P < 0.001 versus control group.

Fig 4: miR-298-5p and miR-100-5p enter microglia, co-localize to their endosomes, and bind directly to human TLR8. a C57BL/6 microglia were incubated with 40 µg/ml pHrodo Red Dextran serving as endosomal marker for 20 min. Subsequently, microglia were exposed to 5 µg/ml of Alexa488-labeled miR-298-5p, Alexa488-labeled miR-100-5p, or Alexa488-labeled let-7b-5p for 4 h. Cells were then fixed and stained with DAPI. b In parallel, microglia exposed to Alexa488-labeled miR-298-5p or Alexa488-labeled miR-100-5p, as described above, were fixed and immunolabeled with TLR7 antibody. (a, b) Cells were analyzed by confocal microscopy with sequential analysis. Representative images of microglia incubated with the indicated fluorescent miRNAs (488 nm, green), stained with DAPI (405 nm, blue), and pHrodo Red Dextran or TLR7 (552 nm, red) are depicted. Green lines indicate region of interest (ROI); scale bar, 10 µm. Representative 3D-vertical slice images of microglia show co-localization of miRNAs and endosomes through different cellular levels (left panel). Diagrams depict fluorescence intensities of the marked ROI in microglia for the sequential analysis used (DAPI: blue line; pHrodo Red Dextran/TLR7: red line; fluorescent miRNA: green line, right panel). (c) Binding affinity measurements of purified polyhistidine-tagged human TLR8 protein and synthetic miRNAs, as indicated, using microscale thermophoresis (MST). TLR8-RNA interaction was monitored by titrating indicated miRNAs from 500 µM to 30 nM against 50 nM RED-tris-NTA-labeled hTLR8 protein and measured with the NanoTemper Monolith NT.115 MST device. Kd values were calculated from dose response curves, which were generated from titration experiments. Data are expressed as mean ± SD, n = 4

Fig 5: Morphine-induced microglial activation involves activation of TLR7. (A) Representative western blot and quantification of Iba1 in the lysates from the thalamus of WT mice administrated saline or morphine (n = 4/group). (B) Representative western blot and quantification of Iba1 in the thalamus lysates of TLR7 KO mice administrated saline or morphine (n = 4/group). (C) Representative images of Iba-1+ cells and pairwise outline shapes were used for morphological parameters measures in the thalamus of WT or TLR7 KO mice administrated saline or morphine. (D) Quantification of morphological parameters: fractal dimension, density, area, perimeter, span ratio and lacunarity in the thalamus of WT and TLR7 KO mice administrated saline or morphine (5 or 6 cells/animal). (E) Real-time PCR analysis of IL-6 /TNFa mRNA expression in adult microglial cells isolated from the brains of TLR7 KO mice administrated morphine (n = 4). (F) Real-time PCR analysis of IL-6 /TNFa mRNA expression in adult microglial cells isolated from the brains of mice administrated EV-anti-miR138 followed by Ctrl-ADEV/Morphine-ADEVs (n = 4). All data are presented as mean ± SD or SEM of four independent experiments. *,P < 0.05; **,P < 0.01; ***,P < 0.001 versus saline group, ###,P < 0.001 versus morphine-ADEV group.