Systemic studies on bacteria- and mammalian cell-derived exosomes

Abstract

Cells communicate and exchange information by several different mechanisms in their environment. Communication between cells is mediated by

(1) secreted growth factors, cytokines, chemokines, hormones and small molecular mediators (e.g., nucleotides, nitric oxide ions, bioactive lipids), (2) direct cell-to-cell contact (through the recently proposed mechanisms of nibbling, trogocytosis and tunneling nanotubes derived from membrane fusion or membrane bridges at the site of intercellular contact). Recent works have emphasized other mechanisms by which cells communicate, e.g., exosomes released by one cell, which transmit essential information to target cells. Exosomes are spherical structures that are limited by a lipid bilayer (of similar structures to that of cell membranes) and that contain hydrophilic soluble components, nucleic acid, and carbohydrates. Exosomes from different cellular origins sequester a common set of molecules that are essential for their biogenesis, structure and trafficking, as well as cell-type specific components which reflect the biological function of the original cells. Various eukaryotic cells release exosomes under physiological or pathological conditions. This phenomenon conserved during evolution, as prokaryote are described to release exosomes (outer membrane vesicles) that are important components for delivery of a signal critical for coordinating group behaviors in bacteria.Colorectal cancer (CRC) is one of the most frequent malignant tumors in Western countries. CRC cells also release exosome, and the revelation from proteomic studies that CRC cell-derived exosomes contain several hundred proteins has helped elucidate the functions of exosomes at the protein level. To improve the understanding of the potential roles of CRC cell-derived exosome in CRC-related processes, from the exosomal and cellular transcripts, I analyzed cellular processes overrepresented by the mRNAs detected in exosomes. Furthermore, I found that CRC cell-derived exosomes enriched with cell cycle-related mRNAs that showed differential expression patterns in CRC patient data. Moreover, exosomes stimulated proliferation of endothelial cells, suggesting that CRC cell-derived exosomes can be involved in tumor growth and metastasis by facilitating angiogenesis-related processes. These results provide a number of indicators that will not only increase the understanding of pathophysiological functions of tumor derived exosomes, but also may stimulate the development of novel methods for cancer diagnostics including CRC. Even though, exosomal protein profiles may have potential use as diagnostic biomarkers of disease, so far, it there are no valuable exosomal biomarkers for CRC metastasis. In order to gain insight the possible metastatic candidate from exosomes, I investigated a metastasis-related differential exosomal analysis between two classic CRC cell lines from the same patient, the primary SW480 and the lymph metastatic SW620. I have identified a total of 850 and 775 unique proteins in SW480 cell- and SW620 cell-derived exosomes, respectively and 276 and 212 proteins were distinctly identified in SW480 cell- and SW620 cell-derived exosomes. Moreover, from the quantification analyses, I found the 2-fold enriched 60 and 87 proteins in SW480 cell- and SW620 cell-derived exosomes, respectively. Among the 2-fold enriched SW620 cell-derived exosomes, MARCKS, HMGA1, VIM and RPSA were involved in tumor invasion and metastasis. Therefore, comparative proteomic approach of exosomes will facilitate our understanding toward the molecular mechanisms of CRC metastasis as well as providing useful biomarkers for cancer prevention, detection and intervention.Tumor cell-derived exosomes expert pleiotrophic roles on tumor microenvironment by transferring exosomal components (e.g., plasma membrane receptors, bioactive lipids, and mRNAs) into target cells, resulting in the modulation of tumor microenvironment for tumor growth, angiogenesis, invasion, metastasis, and immune escape. These multiple functions of tumor-derived exosomes extensively regulate host cells via paracrine mode of action. Given the broad array of bioactive molecules including mRNAs, it would be expected that tumor cell derived-exosomes are not only confined to host cells, including endothelial cells but may also target to neighboring tumor cells themselves. Exosomes isolated from SW480 cells induce proliferation of SW480 cells via ERK-mediated histone H3 phosphorylation, and these inductions were significantly inhibited by the treatment of Wortmanin, U73122, PD98059, and GF10923x, indicating the involvement of PI3K, PLC and PKC signaling pathway on exosomes-mediated cell proliferation. SW480 cell-derived exosomes also induced migration of SW480 cells via p38 MAPK and FAK-mediated actin stress fiber formation. Moreover, tumor cell-derived exosomes were effectively internalized by tumor cells and the amount of exosome uptake by tumor cells was regulated by actin network and PI3K signaling pathway. These results provide the tumor promoting effect of tumor cell-derived exosomes via autocrine stimulation of tumor cell proliferation and migration.A wide variety of Gram-negative bacteria, including Escherichia coli (E. coli), constitutively secrete exosomes, which are defined as spherical, bilayered proteolipids with an average diameter of 20-200 nm. Previous biochemical and proteomic studies have revealed that bacterial exosomes are composed of outer membrane proteins, lipopolysaccharide, outer membrane lipids, periplasmic proteins, DNA, RNA, and other factors associated with virulence. Growing evidence suggests that exosomes released by Gram-negative bacteria play diverse roles in polyspecies communities by enhancing bacterial survival, killing competing bacteria, transferring genetic materials and proteins between bacterial cells, delivering toxins into host cells, and modulating the immune response in host environments. Sepsis, characterized by a generalized inflammatory state caused by infection, is the principal cause of death in hospital populations, and its incidence has increased over the past 20 years. Gram-negative and -positive bacterial infections are the major causes of sepsis, and the syndrome of sepsis develops when the host immune response to infection becomes excessive, which results in systemic inflammation and multiple organ failure. Despite the recent advances in intensive care treatment and the discovery of antibiotics, sepsis remains associated with a high mortality rate. However, strategies to treat sepsis, mainly targeting proinflammaotry mediators, have not been proven to be efficacious in clinical trials. Based on observations of the proinflammatory properties of exosomes derived from E. coli in vitro and in vivo, I investigated the effect of exosomes immunization on systemic inflammation-induced lethality, including sepsis and the detailed protective mechanisms by exosomes immunization. Immunization with exosomes derived from E. coli prevented E. coli-induced lethality and exosomes-induced systemic inflammatory syndrome, accompanied by the involvement of innate and adaptive immune responses triggered by exosome. Moreover, adoptive transfer of splenocytes or T lymphocytes isolated from exosomes-immunized mice protected na？ve recipient mice against E. coli-induced lethality. However, the pre-exposure of exosomes in IFN-r-/- mice did not elicit the protective effect in E. coli-induced lethality, even though exosomes-specific IgG were developed by immunization with exosomes during the immunization periods. These results suggest that exosomes immunization prevents systemic inflammation-induced lethality by induction of innate immunity and CD4+ T cell mediated immune responses through IFN-r secretion.&#8195