Differential traits between microvesicles and exosomes in enterovirus infection

Abstract

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are released by most cell types into the extracellular space and represent the pathophysiological condition of their source cells. Recent studies demonstrate that EVs derived from infected cells and tumors contribute to disease pathogenesis. However, very few studies have rigorously characterized exosomes and microvesicles in infectious diseases. In this study, we focused on subpopulations of EVs during the human enterovirus infection and explored the distinct traits and functions of EVs. We construct an effective immunomagnetic method to isolate exosomes and MVs from enterovirus-infected cells excluding virion. The morphology and sizes of exosomes and MVs have no significant alteration after enterovirus infection. Meanwhile, our study observed that the enterovirus infection could induce exosome secretion but not MVs. In vivo study showed that there was differential biodistribution between exosomes and MVs. Using deep RNA sequencing, we found that the cargo information in MVs rather than in exosomes could accurately reflect pathological condition of original cells. Our study demonstrated that it should be considered to use MVs as clinical diagnostics during in enterovirus infection because their composition is reflective of pathological changes.

Keywords: biodistribution; clinical diagnostics; enterovirus; exosomes; microvesiclesss.

FIGURE 1

Characterization of exosomes and microvesicles from enterovirus‐infected cells. (A) Schematic presentation of isolation procedure to separate microvesicles (MVs), purified exosomes, and virion from CVB3‐infected HCT‐116 cells. (B and C) The size of microvesicles and purified exosomes from mock or CVB3‐infected cells was determined by the nanoparticle tracking analysis (NTA). (D) The electron micrographs of microvesicles and purified exosomes from CVB3‐infected cells were analyzed by Transmission electron microscopy. (E) Purified exosomes and MVs isolated from infected cells above described were determined by western blot using exosomal markers CD9, CD63, and TSG101, MVs markers Annexin A1, Golgi marker GM130 and ER marker calnexin. The lysate of CVB3‐infected cell as positive control.

FIGURE 2

Enterovirus infection induced exosome secretion rather than microvesicles. (A–C) Quantification of exosomes and MVs purified from HCT‐116 cells with different infections was determined using nanoparticle tracking analysis (A and B) and Western blot (C). Data are shown as mean ± SD of four independent experiments. (***p < 0.001). (D and E) The purified exosomes and MVs were isolated from serum of CVB3‐infected, EV71‐infected, or uninfected individuals and quantified using NTA. (n = 6). Data are shown as mean ± SD (**p < 0.01, ***p < 0.001). (F) Schematic presentation of the transwell coculture with HeLa cells in the top well and treatment of HCT‐116 cells in the bottom well. The HCT‐116 cells were cotransfected with CD63‐GFP and Annexin A1‐OFP vectors, and then infected with CVB3 at a 0.05 TCID₅₀ before coculture. A porous (1 μm) membrane allows transfer of exosomes and MVs but precludes direct cell‐cell contact. (G) Fluorescent images of HeLa in the top well were captured at 12 and 18 h after coculture with HCT‐116 cells. Bar = 100 μm. (H and I) Flow cytometric analysis of GFP and OFP ratio in HeLa cells after coculture with HCT‐116 cells at 12 and 18 h. Data are shown as mean ± SD of three independent experiments. (**p < 0.01, ns: nonsignificant).

FIGURE 3

Exosomes and microvesicles exhibited differential biodistribution in vivo. (A and B) The representative IVIS images of different organs were acquired through intravenous injection of exosomes (A, Exo‐EV71, Exo‐CVB3) or MVs (B, MV‐EV71, MV‐CVB3) from enterovirus‐infected cells. Exosomes (Exo‐Mock) or MVs (MV‐Mock) from mock cells as the control. Each mouse was injected with 150 μg DiD‐labeled exosomes or MVs for 24 h, and then animals were euthanized sacrificed for tissue collection. Radiant efficiency was measured using Living Image 3.1 software. (C and D) Fixation of the various organ from mice treated with DiD‐labeled exosomes or MVs for 24 h. After tissue sectioning, representative images were acquired by confocal microscope. The determination of mean gray value (AU) was used Image J software from six different fields.

FIGURE 4

Enterovirus infection did not alter the expression levels of surface proteins for exosomes and microvesicles. (A) Venn diagram of membrane protein in exosomes and MVs though tandem mass spectrometry (LC‐MS/MS). (B and C) The top enrichment of 20 membrane proteins in exosomes and MVs was listed. #PSMs: number of spectrum which protein matched. (D and E) The abundance ratio for top enrichment of 20 membrane proteins in exosomes (D) and MVs (E) from mock and CVB3‐infected cells as determined by LC‐MS/MS.

FIGURE 5

Microvesicle containing protein‐coding RNA profile effectively reflected the pathological response of parental cells. (A–C) KEGG pathway analysis of significantly changed protein‐coding RNAs in CVB3‐infected cells (Cells‐CVB3) as well as cell‐derived exosomes (Exo‐CVB3) or MVs (MV‐CVB3) using DAVID Bioinformatics Resources. HCT‐116 cells were infected with CVB3 for 24 h, then the cells, secreted exosomes, and MVs were collected for RNA‐seq sequencing. Three replicate samples in each group were performed. The KEGG pathway of Cells‐CVB3 vs. Exo‐CVB3 and Cells‐CVB3 vs. MV‐CVB3 were also shown by the Venn diagram (B and C). (D–F) KEGG pathway analysis of significantly changed protein‐coding RNAs in CVB3‐infected cells (Cells‐CVB3), cell‐derived exosomes (Exo‐CVB3), and MVs (MV‐CVB3) using DAVID Bioinformatics Resources. The red font represents the overlapping pathway of Cells‐CVB3 and Exo‐CVB3, while the blue font represents the overlapping pathway of Cells‐CVB3 and MV‐CVB3. (G and H) The significantly changed protein‐coding RNAs of infected cells, derived exosomes, and MVs were analyzed by the GAD_DISEASE tool in the DAVID Bioinformatics Resource. Venn diagrams of known disease of infected cells, exosomes, and MVs were presented. The overlap disease items were listed.

FIGURE 6

The noncoding RNA profile in MVs related to the condition of parental cells. (A and B) Venn diagram of significantly changed miRNAs from CVB3‐infected cells (Cells‐miRNA), cell‐derived MVs (MVs‐miRNA), and exosomes (Exosomes‐miRNA). (C and D) The alteration tendency of changed miRNAs in infected cells, derived exosomes, and MVs according to small RNA‐sequencing data. (E) Venn diagram of significantly changed miRNAs from CVB3‐infected cells derived MVs and exosomes. (F and G) Real‐time PCR analysis of miRNA copy numbers in exosomes and MVs that derived from HCT‐116 cells or Caco‐2 cells. Data are shown as mean ± SD of four independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001).