In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV‐mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd‐sourcing, drawing on the unique EV expertise of academia‐based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

Unmethylated CpG motifs present in bacterial DNA stimulate a rapid and robust innate immune response. Human cell lines and PBMC that recognize CpG DNA express membrane-bound human Toll-like receptor 9 (hTLR9). Cells that are not responsive to CpG DNA become responsive when transfected with hTLR9. Expression of hTLR9 dramatically increases uptake of CpG (but not control) DNA into endocytic vesicles. Upon cell stimulation, hTLR9 and CpG DNA are found in the same endocytic vesicles. Cells expressing hTLR9 are stimulated by CpG motifs that are active in primates but not rodents, suggesting that evolutionary divergence between TLR9 molecules underlies species-specific differences in the recognition of bacterial DNA. These findings indicate that hTLR9 plays a critical role in the CpG DNA-mediated activation of human cells.

Oligodeoxynucleotides (ODN) that contain unmethylated CpG dinucleotides trigger a strong innate immune response in vertebrates. CpG ODN show promise as vaccine adjuvants, anti-allergens, and immunoprotective agents in animal models. Their transition to clinical use requires the identification of motifs that are optimally stimulatory in humans. Analysis of hundreds of novel ODN resulted in the identification and characterization of two structurally distinct “clusters” of immunostimulatory CpG ODN. One cluster (“D”) preferentially stimulates IFN-γ production by NK cells, whereas the other (“K”) stimulates cell proliferation and the production of IL-6 and IgM by monocytes and B cells. The distinct immunostimulatory properties of K and D ODN can improve the design of CpG-based products to achieve specific therapeutic goals.

Bacterial DNA contains immunostimulatory CpG motifs that trigger an innate immune response capable of promoting host survival following infectious challenge. Yet CpG-driven immune activation may also have deleterious consequences, ranging from autoimmune disease to death. We find that repetitive elements present at high frequency in mammalian telomeres, but rare in bacteria, down-regulate CpG-induced immune activation. Suppressive activity correlates with the ability of telomeric TTAGGG repeats to form G-tetrads. Colocalization of CpG DNA with Toll-like receptor 9 in endosomal vesicles is disrupted by these repetitive elements, although cellular binding and uptake remain unchanged. These findings are the first to establish that specific host-derived molecules can down-regulate the innate immune response elicited by a TLR ligand.

Human intestinal flora comprises thousands of bacterial species. Growth and composition of intestinal microbiota is dependent on various parameters, including immune mechanisms, dietary factors and intestinal motility. Patients with spinal cord injury (SCI) frequently display neurogenic bowel dysfunction due to the absence of central nervous system control over the gastrointestinal system. Considering the bowel dysfunction and altered colonic transit time in patients with SCI, we hypothesized the presence of a significant change in the composition of their gut microbiome. The objective of this study was to characterize the gut microbiota in adult SCI patients with different types of bowel dysfunction. We tested our hypothesis on 30 SCI patients (15 upper motor neuron [UMN] bowel syndrome, 15 lower motor neuron [LMN] bowel syndrome) and 10 healthy controls using the 16S rRNA sequencing. Gut microbial patterns were sampled from feces. Independent of study groups, gut microbiota of the participants were dominated by Blautia, Bifidobacterium, Faecalibacterium and Ruminococcus. When we compared all study groups, Roseburia, Pseudobutyrivibrio, Dialister, Marvinbryantia and Megamonas appeared as the genera that were statistically different between groups. In comparison to the healthy group, total bacterial counts of Pseudobutyrivibrio, Dialister and Megamonas genera were significantly lower in UMN bowel dysfunction group. The total bacterial count of Marvinbryantia genus was significantly lower in UMN bowel dysfunction group when compared to the LMN group. Total bacterial counts of Roseburia, Pseudobutyrivibrio and Megamonas genera were significantly lower in LMN bowel dysfunction group when compared to healthy groups. Our results demonstrate for the first time that butyrate-producing members are specifically reduced in SCI patients when compared to healthy subjects. The results of this study would be of interest since to our knowledge, microbiome-associated studies targeting SCI patients are non-existent and the results might help explain possible implications of gut microbiome in SCI.

Synthetic oligodeoxynucleotides (ODN) expressing “CpG motifs” show promise as immune adjuvants, antiallergens, anticancer, and immunoprotective agents. Two structurally distinct classes of CpG ODN have been identified that stimulate human PBMC. This work establishes that both types of ODN bind to and are internalized by the same individual B cells, NK cells, and monocytes. However, the intracellular localization of “D” and “K” ODN differs, as does their functional activity: “K” type ODN trigger monocytes and B cells to proliferate and secrete IL-6 and IgM, whereas “D” type ODN induce NK cells to produce IFN-γ and monocytes to differentiate into CD83+/CD86+ dendritic cells. In monocytes, these two types of ODN (which differ in backbone composition and CpG motif) cross-inhibit one another's activity. Thus, different types of CpG ODN have distinct and in some cases incompatible effects on the same cells, a finding with important implications for the therapeutic use of these agents.