REVIEWS Emerging role of extracellular vesicles in inflammatory diseases Edit I. Buzas, Bence György, György Nagy, András Falus and Steffen Gay Abstract | The discovery that submicron-sized extracellular vesicles (EVs) are generated by both prokaryotic and eukaryotic cells might have a profound effect on experimental and clinical sciences, and could pave the way for new strategies to combat various diseases. EVs are carriers of pathogen-associated and damageassociated molecular patterns, cytokines, autoantigens and tissue-degrading enzymes. In addition to a possible role in the pathogenesis of a number of inflammatory conditions, such as infections and autoimmune diseases, EVs, including microvesicles (also known as microparticles), exosomes and apoptotic vesicles, have therapeutic potential and might be used as biomarkers for inflammatory diseases. Therefore, molecular diagnostics and targeted therapy could benefit from expanding knowledge in the field. In this Review, we summarize important developments and propose that extracellular vesicles could be used as therapeutic vehicles and as targets for the treatment and prevention of inflammatory diseases. Buzas, E. I. et al. Nat. Rev. Rheumatol. advance online publication 18 February 2014; doi:10.1038/nrrheum.2014.19

Introduction Over the past 15 years, advances have been made towards understanding the generation and biological role of cellderived extracellular vesicles (EVs). These membranebound structures occupy a ‘neglected’ size range between molecules and cells. Increasing evidence indicates that EVs contribute to the pathogenesis of various human diseases. In this rapidly progressing field most attention has focused on tumour-derived EVs, and not on the role of EVs in inflammation. In this Review we outline the current knowledge of EVs in i­ nflammatory diseases.

Department of Genetics, Cell and Immunobiology, Semmelweis University, Nagyvárad tér 4, H‑1089 Budapest, Hungary (E.I.B., B.G., A.F.). Department of Rheumatology, Polyclinic of the Hospitaller Brothers of St John of God, Frankel Leó út 54, H‑1023 Budapest, Hungary (G.N.). Centre for Experimental Rheumatology, Clinic of Rheumatology, University Hospital, Gloriastrasse 25, Zürich CH‑8091, Switzerland (S.G.).

Classification EVs is the collective term for cell-secreted phospholipid bilayer-bound structures (Figure 1) that are present in body fluids, are generated by an evolutionarily conserved process, and are secreted by all human cell types tested so far.1–3 EVs are, however, not homogenous and can be classified according to various morphological, biochemical and biogenic parameters (Table 1). Exosomes are EVs that are approximately 50–100 nm in diameter and are generated by the exocytosis of multi­vesicular bodies. EVs that are approximately 100–1,000 nm in diameter and are generated by plasma membrane outward budding and fission1–4 are variably called ‘microvesicles’ or ‘micro­ particles’; no consensus with regard to this nomenclature exists. Historically, clinical flow-cytometry studies have used the term ‘micro­particle’, an ambiguous term that can also refer to nonbiological solid microparticles. The term ‘micro­v esicle’ is less ambiguous and reflects the vesicular nature of these plasma membrane-derived structures. Authors variably designate them as shedding

Correspondence to: E.I.B. [email protected]

Competing interests The authors declare no competing interests.

vesicles, ectosomes or by terms that reflect the source of the microvesicle, such as prostasomes from prostate ­epithelial cells and a­ diposomes from adipocytes. Apoptotic vesicles are EVs that are approximately 100–5,000 nm in diameter and generated by plasma membrane blebbing of cells undergoing apoptosis. Large apoptotic vesicles (1–5 μm) are often referred to as apoptotic bodies.2 Other types of EVs, including large oncosomes5 and retrovirus-like particles, are not discussed in this Review because their role in in­flammatory diseases has not been established.4 Vesicles generated by different biogenic pathways have overlapping size ranges,6 suggesting that neither size nor the mechanism of biogenesis can be used to classify them satisfactorily. Identification and validation of vesicle-specific molecular markers or marker patterns will hopefully enable a general classification in the future. Proposed vesicular markers include DNA or molecular components of cytosolic organelles in apoptotic vesicles, transferrin receptor in exosomes, or vesicle-associated membrane protein 3 and integrin β1 in microvesicles.7 Our knowledge of EVs is substantially based on flow cytometry data, however, depending on the instrument used, the lower size limit of detection with this technique is approximately 300–500 nm.8 Smaller EVs are sometimes detected because of the so-called ‘swarm effect’; a single signal can be generated by many particles travelling together through the cytometer’s flow chamber and laser beam.9 This effect confounds both the enumeration and the size determination of EVs. Size determination is further challenged by the different refractive indexes of vesicles compared with the beads used to calibrate flow cytometers. Serial dilution of flow cytometry samples, standardized devices8 and

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REVIEWS The initiation phase of inflammation

Key points ■■ The evolutionarily conserved release of extracellular vesicles (EVs), including exosomes, microvesicles (microparticles) and apoptotic vesicles, is a previously unappreciated type of secretion by cells ■■ EVs trigger inflammation by carrying pathogen-associated and damageassociated molecular patterns or pathogenic autoantigens ■■ EV-associated cytokines, lipid mediators and microRNAs contribute to the propagation phase of inflammatory diseases ■■ EVs contain proteases and glycosidases that could contribute to tissue destruction ■■ EVs could be used as novel therapeutic vehicles, immunomodulators and therapeutic targets

a

Apo

b Apo

Apo

MV

MV MV Apo Exo Exo

MV

Apo

MV

Exo

Apo

MV

Apo MV

500 nm

500 nm

Figure 1 | Biological samples contain multiple types of extracellular vesicles. Extracellular vesicle populations isolated by centrifugation at 100,000 × g. a | Transmission electron microscopy of conditioned media supernatant from a BV‑2 murine microglial cell line. b | Atomic force microscopy, shaded topography of human blood plasma. Abbreviations: Apo, apoptotic body; Exo, exosome; MV, microvesicle. Image in part a courtesy of Á. Kittel. Image in part b courtesy of M. Csete, M. Deli, A. Szalai and Á. Sipos.

silica beads with a refractive index similar to EVs9 would help to determine EV size. The International Society on Thrombosis and Haemostasis is in the process of addressing the ­s tandardization of EV detection by flow cytometry.10

Functions EVs have many cell-intrinsic functions that are, only now, beginning to be appreciated. Evidence indicates that EVs regulate cell signalling by the removal of plasmamembrane receptors such as during the maturation of reticulocytes,11 the release of signalling molecules,12 the removal or transfer of microRNA (miRNA) 13 and the removal of cytolytic components such as membrane attack complexes.14 EVs also function in cell–cell communication by shuttl­i ng complex messages in the form of membrane proteins, carbohydrates, lipids and other cargo molecules that require protection from extracellular enzymatic degradation, including RNAs, proteins and metabolites. Therefore, EVs can be added to the list of cell–cell communication mediators that includes cytokines, hormones and neurotransmitters.1,2 This intercellular transfer of information combines features of paracrine and juxtacrine (contact-dependent) signalling. Also, vital for host defence against pathogens, EVs are involved in communication between infected cells15 and the i­ nteraction between host cells and bacteria.16

Over the past 30 years, researchers have hypothe­sized that an infectious trigger (through molecular mimi­cry, for example) is involved in the pathogenesis of auto­ immune diseases. The finding that pattern recogni­tion receptors have a critical role in the development of autoimmune diseases (such as rheumatoid arthritis [RA])17 has shed new light on this concept.

PAMPs Gram-negative bacteria secrete EVs (outer membrane vesicles) 18 that contain bacterial antigens and pathogen-associated molecular patterns (PAMPs; Figure 2a). The vesicles secreted by Porphyromonas gingivalis induce mucosal immune responses; 19 how­ ever, their role in triggering RA has not been investigated. In vitro, outer mem­brane vesicles secreted by Helicobacter pylori, Pseudomonas aeruginosa and Neis­seria gonor­rhoeae were shown to deliver peptidoglycan to cytosolic nucleotide-binding oligomerization domain-­containing protein 1 (NOD1) in epithelial cells, leading to upregulation of nuclear factor κB (NF-κB) and NOD1-dependent responses in infected host cells.20 Gram-positive bac­teria also produce EVs; however, they are not referred to as outer membrane vesicles owing to the characteristic lack of an outer membrane. Staphylococcus aureus-derived EVs can induce atopic dermatitis-like skin inflammation 21 and pulmonary inflammation mediated by type 1 helper T (T H1) cells and type 17 helper T (TH17) cells.22 Bacterial EVs have been found to be involved in ­microbial–­host inter­ac­ tions by carrying virulence factors.18 Also, EVs secreted by virulent myco­b acteria have been shown to carry lipoprotein that can be detected by Toll-like receptor (TLR)‑2.23 Bacteria are not the only infectious agents that can release inflammatory EVs. Most (probably all) infectious agents can release inflammatory EVs; for example, the fungus Malassezia sympodialis (formerly known as Pityrosporum) can induce cytokine responses in peripheral blood mononuclear cells from patients with atopic eczema.24 These data suggest that cytokine responses could also be modulated by bacterial EVs in vivo. DAMPs Evidence is accumulating that EVs, carrying damageassociated molecular patterns (DAMPs), released from stressed or injured tissues have a substantial role in the induction and persistence of inflammation (Figure 2a). High mobility group protein B1 (HMGB1), one of the best-studied DAMPs, is highly concentrated within apoptotic vesicles,25 and ATP released from autophagic, dying cells and their phagocytosis activates the inflammasome in macrophages.26 Other EV‑associated DAMPs include heat-shock proteins (HSPs) and S100 proteins, some of which are ligands for TLR2 and TLR4, respectively.17 Despite the association with DAMPs, and the fact that DAMPs have a clear role in the regulation of inflammation, the biological relevance of EV‑associated DAMPs remains to be established.

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REVIEWS Table 1 | Extracellular vesicles Type and size

Biogenesis

Composition

Induction of release

Inhibition of release

Exosomes (50–100 nm)

Exocytosis of multivesicular bodies

MHC molecules and tetraspanins (CD63, CD9, CD81, CD82),1,2,4 Alix, HSPs, TSG101, actin, tubulin, transferrin receptor,7 miRNA and mRNA13

Ionophores,104 calcium heparanase,105 T-cell-receptor activation,106 and P2X purinoreceptor 7107

Proton-pump inhibitors,110 inhibition of neutral sphingomyelinase,111 and rab27a and rab27b silencing114

Microvesicles (100–1,000 nm)

Budding of plasma membrane

Actin and tubulin,2 β1 integrins and VAMP37 and microRNA116

Calcium ionophores,2 thrombin receptor, glycoprotein VI collagen receptor,46 P2X purinoreceptor 7,108 and ARF6 overexpression115

Calpeptin,109 inhibition of ARF6,115 high KCl,112 quinine,112 calpain inhibitor E‑64d,112 and methyl cyclodextrin113

Apoptotic vesicles (100–5,000 nm)

Budding of plasma membrane during apoptosis

Any cellular components, including DNA2 and rRNA116

Inducers of apoptosis

Inhibitors of apoptosis

Abbreviations: ARF6, ADP-ribosylation factor 6; HSPs, heat shock proteins; miRNA, microRNA; rRNA; ribosomal RNA; TSG101, tumour susceptibility gene 101; VAMP3, vesicle-associated membrane protein 3.

Autoantigens One of the most exciting questions related to the role of EVs in inflammation is whether they are targets of autoreactive recognition and thereby capable of triggering or maintaining pathological autoimmune responses. EVs can contain numerous autoantigens implicated in autoimmune diseases, including HSPs, histones, and α‑enolase.27 Importantly, synovial EVs contain citrullinated proteins (including fibrinogen components and CD5 antigen-like precursor, also known as Spα),28,29 which are potential autoantigens involved in the pathogenesis of RA. Immune recognition of EV‑associated antigens has been demonstrated in patients with RA. Large microvesicles in the synovial fluid, for example, were found to form proinflammatory immune complexes with citrullinated protein-specific, vimentin-specific and fibrinogen-specific autoantibodies.28 Data also support a role for EVs as autoantigens in the formation of immune complexes in systemic lupus erythematosus (SLE).30–32 Owing to their DNA and RNA content, EVs might induce autoimmunity by functioning as autoantigens and autoadjuvants that initiate and perpetuate autoantibody production.33 Further­more, salivary gland exosomes have been shown to contain E3 ubiquitin-­protein ligase TRIM21, also known as Ro(SS‑A); Lupus La protein (SS‑B); and the Smith antigen (Sm), which are major autoantigens involved in Sjögren’s syndrome and SLE.34 EVs have also been found in the inflamed joints of patients with juvenile idiopathic arthritis (JIA). 35 A DNA-binding protein involved in chromatin organization, protein DEK, was shown to be secreted by synovial macrophages from patients with JIA, both in a free form and also in exosomes, and was a component of immune complexes. Apoptotic cell death is frequent in physiological and pathological conditions, and it is associated with the release of membrane-bound packages of autoantigens. The immune system has evolved to ‘tolerate’ apoptosis, and autoantigenic components of apoptotic vesicles become accessible for autoimmune recognition only when apoptosis proceeds to an autolytic secondary

necrosis. 36 Caspase‑1-dependent pyroptosis in res­ ponse to a number of bacterial and viral pathogens 37 is associ­ated with a loss of membrane integrity (pore formation) and is inherently proinflammatory. Micro­ vesicles shed from the plasma membrane of pyroptotic cells might have leaky membranes and expose internal autoantigenic components. These data suggest that autoantigens present in EVs are accessible and recognized by autoantibodies in patients with autoimmune diseases (Figure 2b). Recogni­ tion by autoreactive T cells after antigen processing, how­ever, has been investigated only in two studies,38–39 both using nonobese diabetic (NOD) mice. One of these studies showed the activation of autoreactive T cells by ­insulinoma-­derived EVs.38 In the other study, exosomes secreted by islet-derived mesenchymal stem cells carried autoantigens, had potent adjuvant activity and triggered B‑cell and T‑cell autoimmune responses.39

The propagation phase of inflammation Transcellular lipid metabolism Arachidonic acid cascade metabolites, including leuko­ trienes and prostaglandins, can function as potent lipid mediators of inflammation and rheumatic dis­eases.40 The concentrated transcellular delivery of arachidonic acid by platelet-derived EVs, to adjacent platelets and endo­ thelial cells, results in initiation of the cascade, producing thromboxane A2 and cyclo­oxygenase 2,41 demonstrating that EVs can influence the local microenvironment via transcellular lipid metabolism. In addition, macrophagederived and dendritic cell (DC)-derived exosomes have been shown to transfer leuko­triene synthases and cause target cells to produce proinflammatory leukotriene B4 and leukotriene C 4 . 42 Exo­s omes containing active enzymes for leukotriene biosynthesis are also present in plasma from human blood.42 Cytokines EV release is emerging as a mechanism for the secre­tion of proteins that lack a signal sequence, includ­ing IL‑1β, IL‑18,43 galectin‑1, galectin‑3 and HMGB1. IL‑1β, an important proinflammatory cytokine, is secreted by three

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REVIEWS Leaky membranes

EVs (PAMPs)

a Bacteria

Pyroptosis TLRs TLRs

APC Apoptosis NLRs EVs (DAMPs)

Parasite

HMGB1

Fungi

c

b

ATP DNA, nucleosomes

HSPs α-enolase

Protein DEK

IL-1β

Microvesicles IL-8

CX3CL1 IL-1β IL-18

Citrullinated proteins

IL-1β

IL-1β

Exosomes IL-1β

Ro/SSA La/SSB Sm APC

Figure 2 | Extracellular vesicles contribute to inflammation. Recognition of PAMPs and DAMPS by APC can cause inflammation via the secretion of proinflammatory cytokines such as IL-1β. a | Pathogens secrete EVs that carry PAMPs and trigger inflammation. Different types of cell death result in release of EVs that contain DAMPs. EVs from pyroptotic cells have leaky membranes and release endogenous danger signals. Apoptotic vesicles contain high concentrations of the alarmin, HMGB1. b | Autoantigens in EVs (yellow circles) are recognized by autoantibodies and form immune complexes. c | Cytokines are associated with EVs. IL‑1β is released by cells as a cleaved active cytokine, secreted by microvesicles from the cell surface, or is released via exosomes. Activation of the P2X purinoreceptor‑7 by ATP causes IL‑1β release from the vesicle lumen into the extracellular space. Abbreviations: APC, antigen presenting cell; DAMPs, damage-associated molecular patterns; EVs, extracellular vesicles; HMGB1, high mobility group Box 1; HSP, heat-shock protein; NLR, NOD-like receptor; PAMPs, pathogen-associated molecular patterns; TLR, Toll-like receptor.

mechanisms. Caspase‑1 and IL‑1β are trans­located into secretory lysosomes that fuse with the plasma membrane, or are secreted by microvesicles from the cell surface, or caspase‑1–IL‑1β complexes can be released in exosomes.44 Interestingly, activation of the P2X purino­receptor 7 of DC‑derived microvesicles causes e­ xtracellular IL‑1β release from the vesicle lumen (Figure 2c).45 Boilard et al.46 demonstrated the presence and abundance of IL‑1β-containing platelet-derived EVs in synovial fluid from patients with RA. These proinflammatory EVs induced cytokine responses from synovial fibroblasts and contributed to joint inflammation. Chemokines are also transported by EVs. CXCL8 (also known as IL‑8) protein and mRNA can be detected in tumour-derived EVs,47 and apoptotic cells might release the active form of CX3CL1 (also known as fractalkine) inside apoptotic vesicles.48

MicroRNAs miRNAs are small, noncoding RNAs that regulate gene expression. They can also serve as agonists of the ­single-stranded RNA-binding TLRs.49 TLR signalling can activate NF‑κB and the secretion of proinflammatory

cytokines, indicating that miRNAs contribute to inflammation. Extracellular let‑7 miRNAs, which are abun­dant in exosomes,50 have been shown to activate TLR7 and con­tribute to neurodegeneration,51 and miRNA‑21 and miR‑29a can induce secretion of TNF and IL‑6 by bind­ing to TLR7 in mice, and to TLR8 in human macrophages.52 Importantly, miRNAs released by tumour cell EVs have been shown to bind to TLRs on surrounding immune cells causing paracrine activation.53 Another aspect of EV‑associated miRNAs that might be of importance, given the effect of rheumatic diseases on vasculature, was the discovery that a discrete pool of miRNAs that can reprogramme endothelial cells upon in­ternalization can be found in platelet-derived microvesicles.54

The effector phase of inflammation Irreversible tissue damage (cartilage erosion) in RA is considered to be the result of synovial fibroblasts secreting an array of matrix-degrading enzymes, several of which have been found in association with EVs (Figure 3). Importantly, vesicles secreted by synovial fibroblasts from patients with RA had aggrecanase activity.55 Further­more, EVs have been shown to carry several

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REVIEWS Synovial fluid

Synovium

Immune complex with associated microvesicles

T cell

Macrophage

Exosomes APC miRNA Plasma cell IL-8 Apoptotic vesicles

Microvesicles (microparticles)

Angiogenesis PMVs

COX-2 and mPGES-1

Fibrin deposition

Cartilage

Capillary

Fibroblast

Cytokines

β glucuronidase Hexosaminidase D MMP9 MMP14 Aggrecanase

ECM

Bone

B

Figure 3 | Extracellular vesicles in the inflamed joint. Synovial fluid contains the known soluble molecules, such as cytokines and antibodies, and also contains phospholipid bilayer-bound vesicles, including exosomes, microvesicles and apoptotic vesicles. EVs can be found in association with immune complexes. All synovial cell types secrete extracellular vesicles. Endothelial gaps in the synovial blood vessels transmit extracellular vesicles. Platelet-derived microvesicles carry IL‑1β, and induce IL‑8 secretion by synovial fibroblasts. They contribute to fibrin deposition and induce angiogenesis. T‑cell-derived and monocyte-derived microvesicles upregulate COX‑2 and mPGES‑1 expression in synovial fibroblasts, and induce the release of MMPs. MMP9, MMP14 and aggrecanase are found in association with microvesicles, as well as glycosidase enzymes such as β glucuronidase and hexosmaninidase D. Abbreviations: APC, antigen presenting cell; COX‑2, cyclooxygenase 2; ECM, extracellular matrix; EVs, extracellular vesicles; miRNA; microRNA; MMP, matrix metalloproteinase; mPGES1, prostaglandin E synthase; PMVs, platelet-derived microvesicles,

matrix metalloproteinases (MMPs),56 and the release of such proteolytic EVs from human macrophages was induced by tobacco smoke.57 Matrix-degrading glycosidases are also efficient at degrading hyaline cartilage, alone and in combination with MMPs.58 Intriguingly, β‑glucuronidase,59 and hexo­saminidase D glycosidase,60 were found in EVs secreted by synovial fibroblasts. Similar to the extra­cellular matrix invasion, in the case of tumour-derived EVs,7 EVs secreted by synovial fibroblasts might contribute to cartilage erosion in patients with RA.

Inflammatory diseases RA and JIA The role of vesicles in RA, particularly platelet-derived microvesicles (PMV), has been substantiated by many studies: more PMVs were detected in patients with RA

than in healthy individuals, and the number of PMVs correlated with the severity of disease;61 and in a contemporary study, both PMVs and leukocyte-derived microvesicles were detected in the synovial fluid of patients with RA (Figure 3). 62 A number of studies focused on microvesicle numbers in plasma and synovial fluid have found elevated PMV counts in the circulation and inflamed joints.46,63,64 Furthermore, PMVs carry proinflammatory cytokines46 and synovial fluid microvesicles can induce coagulation, contributing to fibrin deposition and joint inflammation (Figure 3).62 The abundance of procoagulant microvesicles in patients with RA might contribute to cardiovascular comorbidities, because platelet-derived EVs are procoagulant, proinflammatory activators of platelets,65 and they decrease the protective function of endothelial cells and induce angiogenesis.66 Microvesicles isolated from the synovial fluid of patients with RA have been shown to have profound biological effects, including the promotion of the synthesis of B‑cell-activating factor,67 and increased production of IL‑8, CCL5 (also known as RANTES), CCL2 (also known as MCP1), IL‑6, intercellular adhesion molecule 1 (ICAM‑1) and vascular endothelial growth factor A (VEGF) by cultured synoviocytes.68 However, in these studies, microvesicles were collected from synovial fluid samples by centrifugation. Subsequent studies showed that microvesicle pellets prepared in this way also contain protein aggregates, immunglobulins and complement proteins.69 The risk of contamination of EVs by protein aggregates was reduced in studies that focused on the effect of leukocyte-derived microvesicles on synoviocytes. Microvesicles produced by platelets were also found to stimulate synoviocytes (via IL‑1β).46 T‑cell-derived and monocyte-derived microvesicles can activate multiple signalling pathways in synovial fibroblasts including NF‑κB, activator protein 1 (AP‑1), p38 mitogen-activated protein kinases and c‑Jun N‑terminal kinase (JNK) pathways.70 Upon activation, synovial fibroblasts upregulated cyclooxygenase 2 and prostaglandin E synthase,70 induced the release of MMPs,71 and increased the production of proangiogenic chemokines.72 In summary, microvesicles derived from platelets and leukocytes in the synovial fluid of patients with RA have a crucial role in inflammation and local joint destruction by activating synovial fibroblasts. Further­more, the enhanced secretion of EVs in RA might increase the risk of cardiovascular diseases. From a biomarker pers­ pec­tive, synovial microvesicle profiles are characteristic for RA; the number of CD3+CD8+ T‑cell-derived microvesicles is significantly increased in patients with RA compared to patients with OA or JIA.64 Theoretically, the outcome of undifferentiated arthritis could be predicted by analysing biomarkers, including synovial fluid microvesicles.

SLE and systemic sclerosis Several reports show higher PMV counts in patients with SLE compared with healthy individuals,73,74 however, in contrast to RA studies, no correlation with disease severity was found. Sellam et al.63 reported a negative

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REVIEWS correlation between PMV counts and the abundance of anti-DNA antibodies. This finding might be the result of increased secretory phospholipase activity, which leads to the destruction of circulating EVs, in severe disease.63 The abundance of EVs in the plasma of patients with SLE might contribute to increased thrombosis and cardiovascular risk. Interestingly, microvesicles from patients with SLE are characterized by a unique protein signature that differentiates them from EVs in patients with RA, systemic sclerosis (SSc) and healthy indivi­ duals.74 Compared with microvesicles from patients with RA or healthy individuals, fewer microvesicles in patients with SLE have a composition that is characteristic of microvesicles from healthy individuals, and more are tagged for removal by immunoglobulins, complement, and other opsonizing molecules.74 This phenomenon could be associated with the defective clearance of apop­ totic bodies that is characteristic of SLE. How­ever, one has to be cautious when interpreting such data. Pro­tein aggregates, such as immune complexes, can be mistaken for microvesicles during flow cytometry or nano­particle tracking analysis. Nonvesicular particles can lead to overestimated microvesicle counts, unless they are distinguished, for example, by their higher resistance to detergent lysis.69 In patients with SLE, microvesicles can contain DNA and react to anti-DNA antibodies.32,75 The mechanism of EV contribution to inflammation and SLE pathogenesis is likely to be similar in RA patho­ genesis. Compared with healthy indivi­duals, endothelial microvesicles are also more abundant in patients with active SLE,76 and can be used as markers of endothelial dysfunction. Treatment of these patients with immuno­ suppressive agents reduced the number of endothelial microvesicles in the ­c irculation and improved ­endothelial function. EVs have also been implicated in SSc. Increased numbers of circulating EVs in patients with SSc suggest that EV‑mediated interaction between activated cell popu­lations contributes to pathogenesis.77 Endothelial cell apoptosis, which has been suggested to be a pri­ mary patho­genic event in SSc,78 might be substantially ­modulated by high concentrations of circulating EVs.79

Therapeutics Encapsulation of small-molecule drugs Aside from being promising biomarkers, EVs could also be used as novel patient-derived vehicles containing exogenously packaged molecules that avoid immune responses. Importantly, EVs have been shown to cross biological barriers, including the blood–brain barrier 80 and synovial membranes.81 Inflamed synovial blood vessels have endothelial gaps that could transmit EV‑sized particles, thereby providing an explanation for the accumulation of PMVs in synovial fluid.81 Many approaches to encapsulate therapeutic agents in EVs can be used. Hydrophobic compounds, such as curcumin, or the signal-transducer and activator of transcription 3 (STAT3) inhibitor JSI‑124 were readily internalized by exosomes after mixing these compounds with vesicles.82 Hydrophilic molecules or macromolecules were reported

to be introduced into EVs using electroporation.80 How­ ever, the effectiveness of this method is a matter of debate because extensive aggregation of small interfering RNA (siRNA) during electroporation might lead to substantial overestimation of the amount loaded into EVs. 83 Another approach is to exploit the endogenous sorting machinery of the cell that directs proteins and RNA into EVs. Some RNA species are selectively enriched in EVs. Bolukbasi et al.84 identified a ‘zipcode’-like sequence in the 3' untranslated region (3ʹUTR) of mRNA that targets it to EVs. Furthermore, a miRNA‑1289 binding site was identified in this region, and miRNA‑1289 was shown to be required for sorting of mRNAs into EVs. These data suggest that it is feasible to generate vesicle-directing, signal-containing therapeutic nucleic acids. Another approach for selective enrichment is to fuse a protein to a vesicular protein, which is then directed into EVs. Using this approach, fusion peptides with lysosome-associated membrane protein 2 (LAMP2) have been used to transport rabies virus glycoprotein peptide to exosomes.80 Shen et al.85 found that the N‑terminal acetylation tag and the PIP2-binding domain of proteins that target them into EVs are the most effective plasma-membrane anchors. Finally, viruses have been incorporated into exosomes; therefore, packaging nucleic acid sequences into viruses, such as the adeno-associated virus (AAV), might result in enrichment in the EV fraction 86 and avoidance of immune responses to viral capsid antigens. Several studies have provided evidence that EVs can carry exogenous RNA, including siRNA,87 miRNA,88 and chemically modified miRNAs.89 Vesicle mediated transfer of these RNA molecules can be functional both in vitro and in vivo. EVs containing anti-inflammatory substances are emerging as therapeutic agents. In the circulation, curcumin encapsulated by exosomes has a longer half-life than free curcumin.82 Exosomal, but not liposomal, curcumin increased the survival of mice after ­lipopolysaccharide-induced septic shock.82 Furthermore, exosomal curcumin and exosomal JSI‑124 were transported to the brain after intranasal administration, further indicating that EVs have exceptionally high bioavailability. Curcumin-loaded exosomes were also efficient at reducing symptoms in experimental models of neuroinflammation.90 However, EV‑encapsulated antiinflammatory drugs or biomolecules have not yet been tested as therapies for rheumatic diseases.

Immunoregulation Reports suggest that EVs have immunostimulatory or immunosuppressive activities depending on their cellular origin and target.1 EVs could be used as immunomodulatory therapeutics for inflammatory, autoimmune or hypersensitivity disorders. In mice, thymic exosome-like particles have been shown to induce CD4+CD25+Foxp3+ regulatory T cell differentiation in the peripheral tissues, partly attributable to transforming growth factor‑β (TGF‑β).91 DC-derived exosomes have robust immunmodulatory activity. Immature bone marrow-derived DCs

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REVIEWS treated with IL‑10,92 or transduced with AAV expressing IL‑10,93 have strong immunosuppressive and anti-­ inflammatory properties. DC exosomes suppressed T‑cell proliferation in mixed lymphocyte reactions,93 and inhibited inflammation in disease models, including murine ­collagen-induced arthritis (CIA),93 murine ­keyhole-limpet haemocyanin-induced delayed-type hypersensitivity (DTH)94 and rat trinitrobenzene sulphonic acid-induced colitis.92 Importantly, the effect was not solely owing to the delivery of IL‑10. In the DTH model, exosome function was dependent on MHC class II, CD178 (also known as FasL)95 and co-stimulatory molecules, such as B7.1 and B7.2.94 Membrane integrity was also crucial, given that freeze–thaw cycling abro­gated the effect.93,95 Exosomes secreted by bone marrow-­derived DCs overexpressing TGF‑β1 have similar anti-­inflammatory effects,96 inducing TREG cells and decreasing the number of type 17 T helper (TH17) cells in a mouse model of inflammatory bowel disease; their effect was superior to recombinant TGF‑β1. 96 DCs overexpressing IL‑4 also produced exosomes that were suppressive in a mouse model of DTH.97 This effect was MHC class II restricted, CD95 (also known as Fas)-dependent and FasL-dependent.97 FasL-expressing exosomes from DC were also anti-inflammatory and immunosuppressive in the DTH model.98 Finally, exosomes from indole­amine 2,3-­dioxy­genase 1 (IDO1)-expressing DCs inhibited inflammation in CIA and DTH models; inhibition was partially de­pendent on co-stimulatory molecules.99 Biological fluids might also contain immuno­ suppressive EVs. Plasma from immunized mice contains exosomes that have a therapeutic Fas-dependent and FasL-dependent effect in the DTH model.95 Further­ more, in plasma from healthy individuals, TNF receptor superfamily member 1A-carrying exosome-like vesicles have been identified.100 These vesicles are hypothesized to modulate TNF-mediated inflammation.100 Strong induction of anti-inflammatory cytokines and growth factors occurs when blood is incubated in the presence of CrSO4 glass beads,101 however, the therapeutic potential of EVs from human autologous conditioned serum has not been investigated. Interestingly, prostasomes contribute to the immunosuppressive activity of human seminal plasma by directly inhibiting phagocytosis by macrophages.102 1.

2.

3.

4.

5.

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Finally, EVs from prokaryotes and yeast might serve as novel vaccines and immunmodulatory agents. Bacteroides fragilis outer membrane vesicles carry capsular polysaccharides that, through a TLR2-dependent mechanism, are immunosuppresive and prevented experimental colitis.103 Collectively, these data indicate that EVs have enormous immunomodulatory potential for treating or preventing inflammatory disorders.

Conclusions Developments in the study of EVs challenge currently accepted paradigms of inflammatory disease mechanisms. EVs are ‘nanocarriers’ and protectors of complex biological information. They have the unique ability to transport membrane and cargo molecules, including proteins and RNA, between cells and quickly spread cellular information without the need for cell migration. EVs are potentially vital contributors to inflammation, by carrying autoantigens, danger signals, cytokines, lipid mediators and tissue-degrading enzymes. Although EVs might not be the main mechanism of transporting cytokines, DAMP-containing EVs released by stressed cells could be vital for autoimmunity, autoinflammation and local immunomodulation. In this emerging field, sufficient evidence now exists to indicate that inflammatory diseases could be treated by targeting EV release. A rapidly expanding body of evidence now indicates that EVs have pivotal roles in the initiation, propagation and regulation of inflammatory diseases; however, the precise physiological and pathological functions of these vesicular structures and their potential as biomarkers, therapeutic vehicles and targets in inflammatory disorders, are not fully understood. Addressing these questions will stimulate researchers in the field for years to come. Review criteria We searched for original articles in the MEDLINE and PubMed databases published between 1980 and 2013. The search terms we used were “extracellular vesicles”, “exosomes”, “microvesicles”, “microparticles” and “apoptotic bodies”. All papers identified were Englishlanguage full text papers. We also searched the reference lists of identified articles for further papers.

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