The biology , function , and biomedical applications of exosomes

Abstract

The study of extracellular vesicles (EVs) has the potential to identify unknown cellular and molecular mechanisms in intercellular communication and in organ homeostasis and disease. Exosomes, with an average diameter of ~100 nanometers, are a subset of EVs. The biogenesis of exosomes involves their origin in endosomes, and subsequent interactions with other intracellular vesicles and organelles generate the final content of the exosomes. Their diverse constituents include nucleic acids, proteins, lipids, amino acids, and metabolites, which can reflect their cell of origin. In various diseases, exosomes offer a window into altered cellular or tissue states, and their detection in biological fluids potentially offers a multicomponent diagnostic readout. The efficient exchange of cellular components through exosomes can inform their applied use in designing exosome-based therapeutics.

Fig. 1.. Identity and the heterogeneity of extracellular vesicles and exosomes.

The two major categories of EVs are ectosomes and exosomes. Ectosomes are released through plasma membrane budding and are in the size range of ~50 nm to 1 μm. Exosomes originate from the endosomal pathway by the formation of the ESEs, LSEs, and ultimately MVBs, which contain ILVs. When MVBs fuse with the plasma membrane, exosomes are released (size range ~40 to 160 nm). Exosomes can be a highly heterogeneous population and have distinct abilities to induce a complex biological response. The heterogeneity of exosomes may be conceptualized on the basis of their size, content (cargo), functional impact on recipient cells, and cell of origin (source). Distinct combinations of these characteristics give rise to a complex heterogeneity of exosomes.

Fig. 2.. Biogenesis and identification of exosomes.

Fluid and extracellular constituents such as proteins, lipids, metabolites, small molecules, and ions can enter cells, along with cell surface proteins, through endocytosis and plasma membrane invagination. The resulting plasma membrane bud formation in the luminal side of the cell presents with outside-in plasma membrane orientation. This budding process leads to the formation of ESEs or possible fusion of the bud with ESEs preformed by the constituents of the endoplasmic reticulum (ER), trans-Golgi network (TGN), and mitochondria. The ESEs could also fuse with the ER and TGN, possibly explaining how the endocytic cargo reaches them. Some of the ESEs can therefore contain membrane and luminal constituents that can represent diverse origins. ESEs give rise to LSEs. Second invagination in the LSE leads to the generation of ILVs, and this step can lead to further modification of the cargo of the future exosomes, with cytoplasmic constituents entering the newly forming ILV. As part of the formation of ILVs, proteins (that were originally on the cell surface) could be distinctly distributed among ILVs. Depending on the invagination volume, the process could give rise to ILVs of different sizes with distinct content. LSEs give rise to MVBs with defined collection of ILVs (future exosomes). MVBs can fuse with autophagosomes, and ultimately the contents can undergo degradation in the lysosomes. The degradation products could be recycled by the cells. MVBs can also directly fuse with lysosomes for degradation. MVBs that do not follow this trajectory can be transported to the plasma membrane through the cytoskeletal and microtubule network of the cell and dock on the luminal side of the plasma membrane with the help of MVB-docking proteins. Exocytosis follows and results in the release of the exosomes with a similar lipid bilayer orientation as the plasma membrane. Several proteins are implicated in exosome biogenesis and include Rab GTPases, ESCRT proteins (see text), as well as others that are also used as markers for exosomes (CD9, CD81, CD63, flotillin, TSG101, ceramide, and Alix). Exosome surface proteins include tetraspanins, integrins, immunomodulatory proteins, and more. Exosomes can contain different types of cell surface proteins, intracellular protein, RNA, DNA, amino acids, and metabolites.

Fig. 3.. Cellular journey of internalized exosomes and endogenously produced exosomes.

Exosomes may directly enter cells by different mechanisms (red). Exosomes are generated de novo by cells through the endocytosis process (blue). Exosomes are continuously being generated by and taken up by cells. It is likely that they can be secreted as a mixture of the de novo-generated and consumed exosomes (red and blue). It is unknown if the release of endogenously generated or consumed exosomes occurs together or separately. Exosomes that are taken up can get degraded by lysosomes. Exosomes that enter cells may enter or fuse with preexisting ESEs and subsequently disintegrate and release their contents into the cytoplasm. Alternatively, endosomes could fuse back with the plasma membrane and release exosomes outside the cells.

Fig. 4.. Exosomes in viral infection.

Exosomes can limit or promote viral infection. Exosomal cargo such as IFNα or APOBEC3G can suppress infection by limiting viral replication or enhancing antiviral immunity. Viruses can also highjack the exosome biogenesis machinery to promote viral dissemination. Exosomes may serve as a pseudoenvelope that enhances viral entry by tetraspanins (CD81, CD9) and PtSer interaction and uptake into recipient cells and aid in evading antiviral immunity. Cotransport of a viral component (proteins and miRNA) may also enhance infectivity. Exosome-mediated transfer of viruses may participate in viral genetic cooperativity and multiplicity of infection. CMV, cytomegalovirus; HSV-1, herpes simplex virus 1.

Fig. 5.. Regulation of immune response by exosomes.

Exosomes from distinct cellular sources, including immune cells (B cells and dendritic cells), cancer cells, epithelial, and mesenchymal cells, shed exosomes with cargos that can influence the proliferation and respective activity of recipient cells of both the innate and adaptive immune system. CD4⁺ and CD8⁺ T cells [cytotoxic T cells (CTL)] can be directly or indirectly influenced by exosomes, stimulating or suppressing their proliferation and function(s). PBMC, peripheral blood mononuclear cell; X?, other potential immunomodulatory proteins.

Fig. 6.. Cellular uptake of therapeutic exosomes.

Therapeutic exosomes isolated from dendritic cells, fibroblasts, and mesenchymal cells can impart specific effects on the target cells, including neoantigen presentation, immunomodulation, and drug payload delivery. The impact of therapeutic exosomes on target cells may be controlled by the different mechanisms of entry or interaction. Entry of intact exosomes can involve receptor-mediated endocytosis, clathrin-coated pits, lipid rafts, phagocytosis, caveolae, and macropinocytosis. Entry of the content of the exosomes, or induction of signals by exosomes, can involve ligand-receptor-induced intracellular signaling or fusion to deposit the contents of the exosomes into the cytoplasm. Examples of therapeutic payload are listed. Target cells include cancer cells, injured parenchymal cells, and immune cells. ASO, antisense oligonucleotide (a DNA oligo-binding RNA target).