Aa EV-induced proinflammatory cytokine expression is blocked in <i>MyD88</i>^<i>-/-</i> mice.

DiD-labeled Aa EVs (approximately 2.25 × 109 particles in 50 μl of PBS) were gingivally injected in WT and MyD88-/- mice after ligature-induced necrosis (n = 6 per each group). (A) 3D rendering of confocal fluorescence image analysis revealed dramatic reduction of proinflammatory cytokines TNF-α (red) and IL-6 (green) by immunostaining (cerebral cortex area) and ELISA (B and C) in MyD88-/- after 24 h of injection (A section: Scale bar = 80 μm). (D) Aa-20050 in the brain was measured using qRT-PCR following intragingival injection of Aa EVs. For qRT-PCR, Cel-miR-39-3p was added (spike-in) for normalization. Data are presented as mean ± SD from six independent experiments. (E) Brain tissues were analyzed by western blotting in response to Aa EVs. NF-κB activation (phospho-p65, upper panel) was significantly decreased in the MyD88-/- mice compared with WT littermate control group at 24 h. Total NF-κB p65 (middle panel) and actin (bottom panel) levels were assessed for loading controls.