外泌體:生物學，功能和在自身免疫性疾病中的新興機會

徐敏雯，侯志平，王烈峰(贛南醫學院．第一附屬醫院; ．基礎醫學院，江西　贛州　34000)

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外泌體:生物學，功能和在自身免疫性疾病中的新興機會

徐敏雯1，侯志平2，王烈峰2
(贛南醫學院1．第一附屬醫院; 2．基礎醫學院，江西贛州341000)

摘要:外泌體是細胞來源囊泡，存在于體內絕大多數體液中。外泌體含有各種分子組分，包括蛋白，RNA與脂質，可由所有培養細胞分泌。該囊泡所攜帶物質賦予其抗原遞呈，細胞通訊，生物標記和免疫調節功能。作者總結外泌體的生物學效用及在自身免疫性疾病中的潛在臨床應用。

關鍵詞:外泌體;生物學功能;自身免疫性疾病;抗原遞呈;免疫調節;多發性硬化;類風濕性關節炎

0　Introduction

Exosomes are small vesicles that contain proteins，RNA，and lipids．They can be secreted by all types of cells in culture and found in most body fluids．Huge efforts have been made to understand their biology and potential clinical use in various diseases including autoimmune diseases．

The secretion of microvesicles (30～100 nm) containing 5＇-nucleotidase activity was initially reported in 1981 from cultured neoplastic cell lines［1］．A few years later，C．Turbide et al．described the release of small vesicles by cultured reticulocytes［2］．Since then，microvesicles have been isolated from nearly all types of mammalian cells，including Schwann cell［3］，adipose cells［4］，fibroblast (NIH 3T3)［5］，antigen-presenting cell (APC)［6］，tumor cell line，and living cells of haematopoietic origin，such as T lymphocytes，B cells［7］，dendritic cells (DCs)［8］，mastocytes，and platelets［9］．In vivo，exosomes are found in most body fluids，including blood［10］，urine［11］，saliva［12］，milk［13］，seminal fluid［14］，amniotic fluid［15］，and carcinomatous effusions［16］．

In the reticulocyte，as in most mammalian cells，portions of the plasma membrane are regularly internalized as endosomes．In turn，parts of the membranes of some endosomes are subsequently internalized as smaller vesicles．Such endosomes are called multivesicular bodies because of their appearance with many small vesicles，or " intralumenal endosomal vesicles" inside the larger body．The intralumenal endosomal vesicles become exosomes if the multivesicular body merges with the cell membrane and release the internal vesicles into the extracellular space［17］．

1　Composition of exosomes

Exosomes contain various molecular constituents of their cell of origin，including proteins，RNA，and lipids．Although the exosomal protein composition varies with the cell and tissue of origin，most exosomes contain an evolutionary-conserved common set of protein molecules．They are cytosolic proteins including tubulin，actin，and actin-binding proteins［18］．They also include various proteins that are involved in metabolism (such as peroxidase，pyruvate kinases，and lipid kinases)［19］and in signal transduction (such as protein kinases and G proteins)［20］．Heat shock proteins (such as HSP70 and HSP90)［21］and MHC class I［22］molecules are also found in exosomes from most cell types．Most of all，several members of Tetraspanins family，such as CD9，CD63，CD81，and CD82，are found in exosomes［23］．It is interesting that all of exosomal proteins，which are related to cell movement，gathering，shaping，and combination，are located in the cytosol，plasma membrane or membrane of endocytic compartments，but not in nuclear，endoplasmic reticulum，Golgi apparatus or mitochondria．Therefore it indicates that the source of proteins is rather limited．

Exosomes also contain proteins with various cell functions．CD86，an important co-stimulatory molecule for T cells，is found in DCs-derived exosomes．T-cell receptors are very abundant in exosomes from T cells［24］．Immunoglobulin superfamily members，such as intercellular adhesion molecule 1，are contained in exosomes from B cells［25］．

The general lipid composition of exosomes is similar to that of the original cell plasma membranes．Phosphatidic acid is enriched at the surface of B-cell-derived exosomes and phosphatidylserine (PS) is found in exosomes from platelets and DCs［26］．

The fact that exosomes can carry mRNA and miRNA as cargo was firstly discovered by a research group from the University of Gothenburg in Sweden．In that study，the differences between cellular and exosomal mRNA and miRNA content，as well as the functionality of the exosomal mRNA cargo，were described［27］．Exosomes have also been shown to carry double-stranded DNA［28］．

2　Biogenesis of exosomes

Vesicle secretion is a general property of animal cells．Trams et al．firstly defined the secreted vesicles as exosomes，and then described vesicles of other size，which were also secreted by the animal cells．Generally，"microvesicle" is used to describe the larger vesicles that pellet at～10 000 g and exosome is used to describe the smaller vesicles that pellet at～100 000 g．However，it is very difficult to find a significant mechanistic difference between microvesicle and exosomes budding at the plasma membranes．Electron microscopy shows that exosomes are intraluminal vesicles in the multivesicular bodies (MVB) that are secreted into the extracellular environment upon fusion of MVB with the plasma membrane［29］．MVBs are endocytic structures that contain small vesicles formed by the budding of an endosomal membrane into the lumen of the compartment．Ariel Savina et al．found that exosomes release was markedly enhanced by a Na+/H+exchanger，which induced changes in the level of intracellular calcium (Ca2 +)．The increase in intracellular Ca2 +level by a calcium ionophore stimulated exosome secretion．On the other hand，a reduction in intracellular Ca2 +level by a chelator completely eliminated exosome release．The results also indicated that transferrin could stimulate exosome release in a Ca2 +dependent manner［30］．Matias Ostrowski et al．identified five Rab GTPases that increase exosomes secretion in HeLa cells using RNA interference (RNAi) screen．Among these GTPases，Rab27a and Rab27b were found to functionin MVB docking at the plasma membrane．The size of exosomes strongly increased by the silence of Rab27a，whereas exosomes were redistributed towards the perinuclear region upon the silence of Rab27b．Thus，the two Rab27 isoforms have different roles in the exosomal pathway．In addition，the silence of two known Rab27 effectors，Slp4 (synaptotagmin-like 4) and Slac2b (exophilin 5)，inhibited exosome secretion and phenocopied the silence of Rab27a and Rab27b，respectively［31］．

The other researchers also reported that the syndecan heparin sulphate proteoglycans and their cytoplasmic adaptor syntenin regulate the biogenesis of exosomes．Syntenin interacts directly with ALIX through LYPX (n) L motifs，similarly to retroviral proteins．Syntenin exosomes depend on the availability of heparin sulphate，syndecans，ALIX and ESCRTs，and impact on the trafficking and connection of FGF signals．This study revealed a key role for syndecan-syntenin-ALIX in the formation of exosomes．Still，the details in exosome biogenesis remain unclear［32］．

3　Function of exosomes

3．1 Antigen presentation

Most of the exosome studies are on the APCs．Raposo et al．showed that exosomes from EBV-transformed B cells stimulated human CD4+T-cell clones in an antigen-specific manner．It was the first to report that exosomes were involved in the release of intact MCH II［33］．Subsequent studies showed that exosomes could also present antigens from tumor cells to DCs．Like exosomes from DCs，exosomes from tumor cells carry MHC II molecules along with tumor antigens that can be recognized by T cells［34］．In conclusion，these studies implicate that exosomes can present tumor antigens to sensitize T cells．

3．2 Cell communication

Several studies have analyzed the biological activities of exosomes in vitro．The researchers found that exosomes could bear surface receptors/ligands interaction，similarly to what happens during the interaction between two cells．And exosomes could bind to target cell membranes，fuse with target cells，and mediate the exchange of membrane and cytosol proteins between two cell types．

Hadi Valadi et al．identified a new cell-cell communication through the exchange of RNA by exosomes，in addition to chemical receptor-mediated events such as direct cell-cell contact and cell-cell synapses．The essential communication between cells is genetic communication，which may occur in the microenvironment．Since exosomes can be trafficked through the systemic circulation as hormones，the communication can occur at a long distance．Exosomes may be more effective in affecting a recipient cell if they deliver a specific mRNA that can modify protein production or miRNA that can modify gene expression of the recipient cell［35］．This makes exosomes an ideal vector for gene therapy because of their ability to deliver nucleic acids to cells at a long distance．

3．3 Biomarkers

The molecular content of exosomes is dependent on the cell type of origin．These molecules are released into extracellular environment that are easily accessed by body fluids such as blood and urine．Therefore，they represent a precious biomedical tool like a fingerprint．For instance，the exosomes of excretion contained a quaporin-2 protein which was a predictive diagnostic biomarker for diabetes insipidus patients［11］．Several studies have suggested that exosomes may be used as biomarkers for the diagnosis and prognosis of malignant tumors，especially for bladder cancer［36］．Some exosome molecules，including epidermal fatty acid binding protein (E-FABP)，migration inhibitor factor-related protein (MRP8)，psoriasin，kertain-14，galectin-7，and stratifin，may be useful for the diagnosis and prognosis of bladder cancer［37］．Tspan8，CD44v6，and alpha6beta4 are biomarkers of migrating pancreatic cancer-initiating cells［38］．Exosomes may also be valuable as biomarkers for other diseases．Similar proteomic studies on urinary exosomes identified multiple molecular markers for diagnosis，prognosis，and pathophysiology［39］．Urinary activating transcription factor 3 (ATF3) and exosomal Fetuin-A might be predictive biomarkers for structural renal injury［40］．Van der Lubbe and colleagues found that the phosphorylated sodium chloride cotransporter in urinary exosomes was superior to prostasin as a biomarker for aldosteronism［41］．

3．4 Immune regulation Function

Exosomes have also been shown to be involved in immune functions．For example，Zhou et al．screened the expression and distribution profiles of immune-related miRNAs of the exosomes in breast milk．The results showed that miR-181a and miR-155 in the exosomes were present at different stages of lactation and might modulate the immune response in infants．They also found that these miRNAs had high stability and were resistant to harsh conditions，such as RNase digestion and extreme temperatures［42］．

Exosomes in tumor cells can also induce an immune reaction．Tumor-derived exosomes usually contain tumor antigens and can be used to stimulate antitumor immune responses．However，many reports also suggest that tumor-derived exosomes can facilitate tumor immune evasion through different mechanisms［43］．

NKG2D，an activating cytotoxicity receptor，plays an important role in immune suppression in cancer．Marie Lundholm et al．investigated whether prostate tumor-derived exosomes can down-regulate NKG2D expression on natural killer (NK) and CD8+T cells［44］．Using flow cytometry，they found that the prostate tumor-derived exosomes selectively induced the downregulation of NKG2D on NK and CD8+T cells in a dose-dependent manner．Consistent with these findings，patients with castration-resistant prostate cancer (CRPC) showed a significant decrease in surface NKG2D expression on circulating NK and CD8+T cells compared to healthy individuals．Incubation of healthy lymphocytes with exosomes from serum or plasma of CRPC patients induced the down-regulation of NKG2D gene expression in lymphocytes．These data suggested that prostate tumor-derived exosomes can down-regulate the NKG2D-mediated cytotoxic response in CRPC patients，and thus promote immune suppression and tumor escape［44］．

Using a mouse model of delayed-type hypersensitivity (DTH)，Yang and colleagues demonstrated that tumor-derived exosomes could induce tumor antigenspecific immune suppression［45］．It was found that，following local injection，tumor-derived exosomes were internalized by CD11c+cells and transported to the draining lymph nodes (LN)．Exosome-mediated suppression of DTH response was associated with increased mRNA levels of TGF-β1 and IL-4 in the draining LN．They also found that tumor-derived exosomes could inhibit DCs maturation．Taken together，tumor-derived exosomes could induce tumor antigen-specific immune suppression［45］．

4　Biological functions of exosomes in autoimmune diseases

Exosomes can carry large amounts of proteins that are related to cell movement，gathering，shaping，combining，and can transfer those molecules to another cell via membrane vesicle trafficking．This indicates that exosomes resemble the biology of cells from which they are derived．Thereby，the exosomes derived from the immune system，such as dendritic cells and T cells，may play a functional role in mediating autoimmune responses．Some experiments have demonstrated that exosomes from immature or suppressive DCs exhibited the capacity to induce immune tolerance in murine models of autoimmune diseases［46］．

4．1 Multiple sclerosis

Multiple sclerosis is an autoimmune disease due to oligodendrocyte loss，demyelination and failure to remyelinate damaged brain areas．Oligodendrocytes produce CNS myelin，the insulation surrounding axons that are necessary for neuronal signaling in learning，memory，and cognition．Remyelination is a spontaneously repair process in which oligodendrocyte precursor cells are recruited to damaged areas and subsequently differentiate into mature oligodendrocytes that are capable of replacing lost myelin［47］．

Current therapies for multiple sclerosis largely focus on reducing demyelination via immune suppression，and often come with an array of harmful immune sequence．Thus，some researchers attach more importance to promoting remyelination as a therapeutic target．They suggest that use of exosomes to stimulate remyelination would be beneficial to multiple sclerosis patients［48］．

Lei Yu and colleagues demonstrated that exosomes from DCs could secret TGF-1 (sTGF-1-EXOs) and de-lay the development of murine inflammatory bowel disease (IBD)．Subsequently，they isolated exosomes from DCs expressing membrane-associated TGF-1 (mTGF-1-EXOs) and found mTGF-1-EXOs had more potent immunosuppressive activity than sTGF-1-EXOs in vitro．mTGF-1-EXOs inhibited the development and progression of experimental autoimmune encephalomyelitis (EAE)，an animal model of multiple sclerosis．More importantly，mTGF-1-EXOs could reverse the disease by impairing Ag-specific Th1 and IL-17 responses and promoting IL-10 responses ex vivo．The reduction of Th17 cell frequency by mTGF-1-EXOs were correlated with the down-regulation of the p38，ERK，Stat3，NF-κB activation，and IL-6 expression in DCs．At the mean time，adoptive transfer of CD4+Foxp3+Treg cells into mTGF-1-EXO-treated EAE mice dramatically prevented the development of EAE in the recipients．Moreover，mTGF-1-EXOs from C57BL/6 mice effectively prevented and inhibited proteolipid protein (PLP ) peptide-induced EAE in BALB/c mice．These results indicate that mTGF-1-EXOs possess powerful immunosuppressive ability and can effectively inhibit the development and progression of EAE in different strains of mice［49］．

Pusic et al．found that serum exosomes promoted the differentiation of oligodendrocyte precursor cell (OPC) into mature myelin-producing cells under control conditions and after acute demyelination．Exosomes from environmental enrichment (EE) of aging animals mimicked this promyelinating effect．They found that both young and EE serum-derived exosomes contained rich miR-219，which is necessary and sufficient for production of myelinating oligodendrocytes by reducing the expression of inhibitory regulators of differentiation．After exosomes were used to slice cultures，the protein transcript levels of these miR-219 target mRNAs were decreased．Nasal administration of exosomes to aging rats also enhanced myelination．Thus，peripheral circulating cells in young or environmentally enriched animals produce exosomes that may be a useful therapy for remyelination［48］．

Some researchers have tried to determine whether the exosomes can function as a delivery vehicle to carry anti-inflammatory drugs．They used the exosomes to encapsulate curcumin (Exo-cru) or a Stat3 inhibitor，JSI124 (Exo-JSI124)．The EAE mice treated intranasally with Exo-cru or Exo-JSI124 had significantly delayed EAE progression．Intranasal administration of Exo-cur or Exo-JSI124 led to rapid delivery of exosome-encapsulated drugs to the brain．The drugs were then selectively taken up by microglial cells，which subsequently induced apoptosis of those microglial cells．The results demonstrated that exosomes might provide a novel therapeutic approach to treat MS［50］．

4．2 Rheumatoid arthritis

Rheumatoid arthritis (RA) is a common，chronic，idiopathic autoimmune inflammatory disease characterized by synovitis，inflammatory joint fluid，degradation of articular cartilage，erosion of the marginal bone，systemic immune，and inflammatory manifestations．The level of clinically useful RA biomarkers can increase due to age，anemia，and the presence of immunoglobulins，suggesting that they may not be ideal for RA diagnosis．Thus we need more biomarkers which can diagnose RA effectively and conveniently．K．Skriner et al．determined the nature of synovial exosomes from patients with different rheumatic diseases and examined their potential autoantigenic content，which may be involved in the induction of an autoimmune response．They found that citrullinated proteins were observed in all exosome preparations，in contrast to other autoantigenic proteins (e．g．，BiP and heterogeneous nuclear RNP A2) that were previously observed in RA and other autoimmune diseases．These citrullinated proteins included the fibrin alpha-chain fragment，fibrin beta-chain，fibrinogen beta-chain precursor，fibrinogen D fragment，and the Sp alpha (CD5 antigen-like protein) receptor．Purification of synovial exosomes led to the detection of citrullinated fibrinogen and citrullinated Sp alpha that were associated with IgM and IgG production．These results suggested that citrullinated peptides carried by exosomes，known as autoantigens in RA，could play an important role in the induction and distribution of citrullinated proteins［51］．

Exosomes from B cells，T cells，dendritic cells (DCs)，and mast cells can confer immunoregulatory signals between cells in either an immunostimulatory or an immunosuppressive manner．Indeed，exosomes fromAPCs contain many important regulatory molecules for antigen-presentation function，such as class MHC I，MHC II，CD80 (B7-1)，and CD86 (B7-2)，as well as various adhesion molecules that may target exosomes to their acceptor cells．S．H．Kim and colleagues demonstrated that stimulation of exosomes from immature DC with immunomodulatory cytokines (i．e．，IL-10 and IL-4) could effectively treat collagen-induced arthritis (CIA)．The DC-derived exosomes were internalized by CD11c (+ ) DC in the dermis at the site of injection and in the draining lymph node as well as by CD11c (+ ) DC and F4/80(+ ) macrophages in the spleen．The results showed that administration of DC/ IL-4 or exosomes derived from DC/IL-4 can regulate the activity of APC and T cells in vivo through a MHC class II and partly Fas ligand/Fas-dependent mechanism．In fact，the exosomes were as effective as the parental DC in RA treatment．Because purified DC-derived exosomes are very stable vesicles，they represent a better approach than unstable DC for future treatment of arthritis and other autoimmune disorders［46］．

A tryptophan-degrading enzyme，indoleamine 2，3-dioxygenase (IDO) is important to regulate immune responses and maintain tolerance．Bianco and his colleagues examined the immunosuppressive effects of exosomes from IDO-positive DCs and found that IDO overexpression in both DCs and DC exosomes had an anti-inflammatory effect in murine CIA models．The suppressive effects were partially dependent on B7 costimulatory molecules．Therefore，exosomes from IDO-positive DCs may represent a novel therapy for RA［52］．

Martinez-Lostao，Luis et al．previously observed that T lymphocytes in synovial fluid (SF) of patients with RA were sensitive to APO2L/TRAIL treatment．In addition，there was a drastic decrease in the amount of bioactive APO2L/TRAIL within exosomes in SF from RA patients．They used artificial lipid vesicles (liposomes) to resemble natural exosomes．APO2L/TRAIL bound to liposomes was intra-articularly injected into the knees of animals with AIA (antigen-inducedarthritis)，and substantially reduced synovial hyperplasia and inflammation in rabbit knee joints．It suggested that the increased bioactivity of APO2L/TRAIL associated with liposomes was due to the increase in the local concentration of the recombinant protein，augmenting its receptor crosslinking potential，instead of conformational changes in the protein［53］．

4．3 Others autoimmune disease

In addition，the function of exosomes in other autoimmune diseases has been studied extensively．Schilders，Geurt and colleagues demonstrated that a recently identified exosome-associated protein，C1D，is a major autoantigen in patients with the PM-scleroderma overlap syndrome，and suggested that the use of recombinant C1D as an autoantibody target may aid in the diagnosis of this disease［54］．Salama and Apolline et al．found that endogenous miR-29b，spontaneously released from exosomes within beta-cells，could induce TNFα secretion from spleen cells in mice with autoimmune diabetes in vitro［55］．

5　Perspectives

A lack of understanding on the cause of autoimmune disease，as well as effective means of disease diagnosis，treatment，and prevention，make it unlikely that one single biomarker or drug will satisfy the needs of autoimmune disease．Researchers and doctors all over the world have been struggling for decades to find a better way for diagnosis，treatment，and prevention of autoimmune disease．The utilization of exosomes might provide a better choice and continued progress in exosomes research will definitely lead to more extensive clinical application in the treatment of some autoimmune diseases．

Abbreviations: MVB: multivesicular bodies，NK: natural killer，MS: Multiple sclerosis，RA: Rheumatoid arthritis，APC: antigen-presenting cell，DCs: dendritic cells，EAE: experimental autoimmune encephalomyelitis

Competing interest: All authors declared no conflicts of interest．

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(To be continued on p12)