FIG 1.

TIM-1 mediates exosome internalization and IFN-α-induced anti-HBV activity transmission. (A) Electron microscopy of purified exosomes from macrophages. Scale bar: 100 nm. (B) Immunoblot analysis of macrophage-derived exosomes (left) and corresponding cells (right) for exosomal and nonexosomal markers. (C) PKH26-labeled exosome internalization by HepG2 cells. Scale bar: 5 µm. (D) PtdSer detection on the macrophage exosome surface. Exosomes coating 4-µm latex beads were either stained or not with annexin V-FITC and analyzed by flow cytometry. (E) Knockdown validation of TIM-1 by immunoblotting. (F and G) Confocal images (F) or flow cytometry analysis (G) of PKH26-labeled exosome internalization by HepG2 cells after TIM-1 knockdown. Scale bars: 10 µm. For flow cytometry analysis, both histogram graph (left) and mean fluorescence intensities (MFI) (right) normalized to control siRNA (siCTRL)-transfected cells are presented. (H) Flow cytometry analysis of PKH26-labeled exosome internalization by HepG2 cells in the presence or absence (Ctrl) of Fc-TIM-1-His. MFI (right) is normalized to that of Ctrl cells. (I) Binding of GFP-exosomes preincubated with or without Fc-TIM-1-His to HepG2 cells with plasma membrane stained by PKH26. Scale bars: 5 µm. (J) PtdSer detection on the surfaces of exosomes derived from HepG2, LX-2, MGC-803, and HEK293T cells. (K) Flow cytometry analysis of PKH26-labeled HepG2 or LX-2 exosome internalization by HepG2 cells after TIM-1 knockdown. (L) Comparison of TIM-1 expression among HepG2, LX-2, and MGC-803 cells by immunoblotting. The expression of TIM-1 was normalized against β-actin and is presented (as percent) relative to its expression in HepG2 cells. (M) Detection of TIM-1 (brown staining) in normal human liver section by IHC. (N) Flow cytometry analysis of PKH26-labeled macrophage exosome internalization by LX-2 or MGC-803 cells after TIM-1 knockdown. The TIM-1 knockdowns were validated by immunoblotting. (O) HepG2.2.15 cells were transfected with either siTIM-1 or siCTRL. The level of HBsAg in the culture medium (supernatant) was detected by ELISA. HBV DNA in the culture medium and intracellular core particle DNA were quantified by qPCR. (P) Blockade of IFN-α-induced anti-HBV activity transmission by TIM-1 knockdown. HepG2.2.15 cells transfected with either siTIM-1 or siCTRL were treated with exosomes from IFN-α-stimulated macrophages (IFN-EXO) or unstimulated cells (Ctrl-EXO). HBsAg and HBV DNA levels in the medium were measured by ELISA or quantified by qPCR. The error bars indicate the SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student’s t test). The data are representative of those from three independent experiments.