Extracellular vesicles from infected cells: potential for direct pathogenesis

Abstract

Infections that result in natural or manmade spread of lethal biological agents are a concern and require national and focused preparedness. In this manuscript, as part of an early diagnostics and pathogen treatment strategy, we have focused on extracellular vesicles (EVs) that arise following infections. Although the field of biodefense does not currently have a rich resource in EVs literature, none the less, similar pathogens belonging to the more classical emerging and non-emerging diseases have been studied in their EV/exosomal contents and function. These exosomes are formed in late endosomes and released from the cell membrane in almost every cell type in vivo. These vesicles contain proteins, RNA, and lipids from the cells they originate from and function in development, signal transduction, cell survival, and transfer of infectious material. The current review focuses on how different forms of infection exploit the exosomal pathway and how exosomes can be exploited artificially to treat infection and disease and potentially also be used as a source of vaccine. Virally-infected cells can secrete viral as well as cellular proteins and RNA in exosomes, allowing viruses to cause latent infection and spread of miRNA to nearby cells prior to a subsequent infection. In addition to virally-infected host cells, bacteria, protozoa, and fungi can all release small vesicles that contain pathogen-associated molecular patterns, regulating the neighboring uninfected cells. Examples of exosomes from both virally and bacterially infected cells point toward a re-programming network of pathways in the recipient cells. Finally, many of these exosomes contain cytokines and miRNAs that in turn can effect gene expression in the recipient cells through the classical toll-like receptor and NFκB pathway. Therefore, although exosomes do not replicate as an independent entity, they however facilitate movement of infectious material through tissues and may be the cause of many pathologies seen in infected hosts.

Keywords: bacteria; exosome; extracellular vesicle; parasite; pathogen; protozoa; virus.

FIGURE 1

Purification of exosomes using centrifugation or affinity beads. (A) An overview of a generalized procedure utilizing multistep centrifugation for high volume of culture medium. A culture of infected cell supernatants (∼100 ml grown in exosome free media) is centrifuged at multiple speeds and for varying times. The samples are next filtered to remove large debris including dead cells. After the final ultracentrifugation, the sample represents partially purified exosomes (∼80%). Exosomes can be further purified using sucrose density or OptiPrep gradient (>95%). The x-axis represents % iodixanol in OptiPrep gradients. These fractions can then be further purified with magnetic beads coated with antibodies to exosomal proteins such as C63 [usually present in higher amounts from RNA virus infected cells such as HIV, HTLV-1, RVFV and Ebola]. (B) An overview of low volume purification with Nanotrap particles to separate exosomes from the virus particles. Nano-captured exosomes can be used for PCR analysis (modified from Sampey et al., 2014).

FIGURE 2

Exosomes from virally-infected cells have various specific effects on target cells. Exosomes from HIV-1- and HTLV-1-infected cells contain viral and cellular proteins and double- and single-stranded RNA. Within a target cell, the proteins decrease apoptosis and cell migration, whereas RNA increases gene expression in the nucleus in order to promote infection. Exosomes from HCV-infected cells contain non-enveloped virions (both mature and immature). These virions can enter a target cell using exosomes and replicate on the ER membranes. Exosomes from HHV-6-infected cells contain mature virions that spread infection faster through the use of exosomes. Exosomes from EBV-infected cells contain viral DNA and viral and cellular proteins that increase cytokine and chemokine production in target cells (through innate immune molecules), and thus increase cytokine expression and inflammation. Exosomes from KSHV-infected cells contain viral miRNAs and proteins that increase glycolysis and gene expression in target cells.