Microvesicles: ubiquitous contributors to infection and immunity.

Epub ahead of print December 3, 2014 - doi:10.1189/jlb.3RU0513-292RR

Review

Microvesicles: ubiquitous contributors to infection and immunity Frances W. Lai, Brian D. Lichty, and Dawn M. E. Bowdish1 McMaster Immunology Research Centre, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada RECEIVED MAY 21, 2013; REVISED AUGUST 26, 2014; ACCEPTED SEPTEMBER 21, 2014. DOI: 10.1189/jlb.3RU0513-292RR

ABSTRACT MVs, which can be subgrouped into exosomes, SVs, and OMVs, are secreted by eukaryotic and prokaryotic cells. Many previously inexplicable phenomena can be explained by the existence of these vesicles, as they appear to be important in a wide range of biologic processes, such as intercellular communication and transfer of functional genetic information. In this review, we discuss the immunologic roles of MVs during sterile insult and infectious disease. MVs contribute to clotting initiation, cell recruitment, and neovascularization during wound healing. In the context of pathogen infection, both the host and the pathogen use MVs for communication and defense. MVs are exploited by various viruses to evade the host immune response and contribute to viral spread. Bacteria produce MVs that contain virulence factors that contribute to disease pathology and antibiotic resistance. This review summarizes the role of MVs in the pathology and resolution of disease J. Leukoc. Biol. 97: 000–000; 2015.

Introduction Discoveries In the 1960s and ’70s, researchers studying prokaryotic and eukaryotic cells independently discovered that small membranes shed from the surface of the cell had diverse biologic effects. Researchers discovered that prokaryotes shed membranous sacs from their cell surfaces, and these contained the LPS that was detected in cell-free Escherichia coli cultures [1, 2]. Pinching off of bacterial cell wall in Vibrio cholerae was first reported in 1967 and offered an explanation as to the mechanism of cholera toxin secretion [2]. Since then, OMVs have been shown to be produced by a multitude of pathogenic and nonpathogenic bacteria and have been found in biofilms, in the broth and agar plates of laboratory cultures, and in infected individuals. In parallel studies in eukaryotic systems, Wolf [3] speculated that particulate matter sedimented from plasma possessed Abbreviations: CFTR = cystic fibrosis transmembrane conductance regulator, Cif = cystic fibrosis transmembrane conductance regulator inhibitory factor, DC = dendritic cell, Del-1 = developmental endothelial locus 1, FGF2 = fibroblast growth factor 2, gB = glycoprotein B, LMP1 = latent membrane protein 1, miRNA = microRNA, MV = microvesicle, MVB = multivesicular body, OMV = outer membrane vesicle, SV = shedding vesicle, TF = tissue factor

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coagulation properties. This “platelet dust,” as he called it, was distinguishable from intact platelets, and he explained why plasma, when stored for long periods of time, began to coagulate. Subsequently, in the early 1980s, groups showed that activated platelets secreted coagulation factors in phospholipid vesicles [4, 5]. Around the same time, the term of exosomes was coined for the vesicles shed from the cell surface [6]. Rose Johnstone’s group [7] at McGill University went on to show that in reticulocytes, these exosomes accumulated in multivesicular structures or bodies and were used to shed selectively the surface protein receptors, namely transferrin. The major ligand for transferrin receptors is iron-bound transferrin, and this finding offered an explanation for the mechanism of iron transfer, as well as how the receptor is lost during reticulocyte maturation [7]. Phenomena such as this, in addition to other observations, such as trogocytosis (the transfer of membrane fragments at immunologic synapse between APC and T cell) and nibbling (capture of antigen from live cells by DCs), can now be explained by the existence and release of cellular MVs. In the 30 years since Johnstone’s discovery, research in this area has exploded, and it is now known that these small cell-derived membrane structures are important messengers in cellular communication. Every cell type examined to date has been shown to secrete some form of MV, and it appears to be a mechanism conserved across all kingdoms from Archaea to Plantae. The past decade has seen a rise in the understanding of the significant role that MVs play in many aspects of biologic function and communication. For example, the observation that small, noncoding RNA, known as miRNA, can be packaged specifically into MVs and that this packaging contributes to its stability in vivo partially explains the phenomena that circulating miRNA can be detected and used as biomarkers of certain diseases [8, 9]. Tumor cells also continuously shed tumor antigen-containing vesicles, which are absorbed by APCs, and circulating vesicles can be used as biomarkers to detect particular cancers [10]. This knowledge also provides us with tools with which to create therapies, such as vaccines and cancer therapeutics [11–15]. This is an extremely important developing area but is beyond the scope of this review.

1. Correspondence: McMaster University, MDCL 2319, 1200 Main St. West, Hamilton, ON, L8N 3Z5, Canada. E-mail: [email protected]; Twitter: http://www.twitter.com/@MsMacrophage

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Copyright 2014 by The Society for Leukocyte Biology.

We refer interested readers to several excellent reviews that have been published on the use of MVs in drug delivery and the development of cancer therapies (see refs. [16–22]).

[28, 29], as they are not technically MVs, we have excluded them from this discussion and will focus on exosomes and SVs.

Nomenclature

MVS AND WOUND HEALING

As the field expands, nomenclature has become increasingly inconsistent, which can cause confusion to neophytes and specialists in the field. To clarify the terms used in the literature, a summary of terminology and characteristics of MVs has been generated (see Table 1). In broad terms, there are 3 differing types of MVs that originate from eukaryotic cells: 1) exosomes, 2) SVs, and 3) apoptotic bodies. The term MV is often used as an umbrella term to encompass categories I and II and will be used as such in this review. The vesicles can be differentiated based on size (exosomes are the smallest; apoptotic bodies the largest) and shape (SVs are irregularly shaped, whereas exosomes are homogeneously cup shaped, as observed by transmission electron microscopy). In addition to size, the origin of vesicles is another chief distinguishing feature. Exosomes are derived from the cellular secretory pathway and accumulate in MVBs before release. SVs are, as the name implies, shed directly from the plasma membrane surface of the cell. In contrast, apoptotic bodies are generated as the cell is undergoing apoptosis. Although apoptotic bodies may resemble MVs, they can be distinguished by their large size and heterogeneous shape. They have been found to contain DNA and tumor antigens and to express phosphatidylserine on the surface [23–25]. Apoptotic bodies tend to elicit an anti-inflammatory or tolerogenic response when taken up by APCs or neighboring cells. However, apoptotic bodies have also been implicated as playing a role in the generation of autoimmunity [26, 27]. Although apoptotic bodies are an important means of cellular communication

Postinsult, the process of wound healing entails clotting, cell recruitment, resolution of inflammation, and neogenesis, respectively. MVs are involved in each step of the process. After an initial insult to the vascular system, MVs play an important role in the clotting process, which is the initial step of healing. Platelet activation, which occurs within minutes, stimulates release of MVs (Fig. 1A) [30]. A multitude of clotting factors has been found to be expressed in MVs released from activated platelets, and these MVs appear to possess more procoagulant activity than the platelets themselves [31]. Platelet MVs, as well as MVs derived from monocytes, have been shown to bear TF, a type I transmembrane protein that initiates blood coagulation (Fig. 1A) [32, 33]. Along with TF, MVs from activated monocytes also contain P-selectin glycoprotein ligand 1, a ligand that binds Pselectin expressed on activated platelets in blood clots and is a mandatory interaction for the continuation of the clotting process (Fig. 1A) [34]. Selective enrichment of TF and P-selectin ligand in cholesterol-rich lipid rafts has been shown to occur in activated monocytes, and this localization, as well as MV secretion, was blocked in cholesterol-depleted cells [33]. Once MVs are secreted and come into contact with activated platelets, protein transfer occurs, and platelets acquire TF and P-selectin ligand from the monocyte-derived MVs [34]. After clotting is initiated, immune cells, such as neutrophils and macrophages, are recruited to the wound site. Activated macrophages and DCs release MVs containing proinflammatory components, such as enzymes that catalyze leukotriene

TABLE 1. Comparison of MVs Vesicle type Exosome

Shedding MV

Apoptotic body

OMV

Apoptotic vesicle

Archaeosomes (archaea [78]), membrane vesicles (Gram-positive)

30–100 [59, 79, 80]

Microparticle (platelets, monocytes), ectosome (neutrophils, fibroblasts [42]), exovesicle (DC [61]) 100–1000 [6, 30]

50–5000 [27]

Luminal markers

1.1–1.21 [59, 79, 80] Cup, homogeneous 1,000,000 [59, 79, 80] Tetraspanins (CD63, CD9), TNFR1 ALIX, TSG101, miRNA

1.14–1.18 [30] Irregular shape, variable 10,000–20,000 [6, 30] CD40 ligand, integrins, MMPs, phosphatidylserine Annexin 2, caspases, FGF2

1.16–1.28 Variable, heterogeneous 1200–100,000 [27] Phosphatidylserine, histones DNA, RNA

10–300 [81, 82] (Gramnegative), 50–150 [83] (Gram-positive) 1.2–1.22 [82, 83] Spheroid, heterogeneous 40,000–100,000 [81, 82] LPS, glycerophospholipids

Site of generation

Multivesicular bodies

Plasma membrane

Mechanism of generation

Budding, exocytosis of multivesicular bodies

Budding of plasma membrane

Plasma membrane, cytoplasm, nuclei Cell shrinkage and death, release from cells undergoing apoptosis

Other terminology (source)

Dexosome (DC [76]), texosome (tumor cells [77])

Size (nm) Density (g/ml) Shape Sedimentation (g) Surface markers

Strain-dependent virulence factors Bacterial outer membrane Budding from outer membrane

ALIX, apoptosis-linked-gene-2-interacting protein X; MMPs, Matrix metalloproteinases; TSG101, tumor susceptibility gene 101.

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Microvesicles in infection and immunity

Figure 1. MVs in wound healing. MVs are involved in each stage of wound healing: (A) clotting, (B) cell recruitment, and (C) resolution of inflammation and neogenesis. (A) Both platelets and monocytes release procoagulant-containing MVs that aid in clot formation. (B) Cells are recruited to the wound site via the release of MVs from DCs, macrophages, and monocytes. These MVs contain leukotriene-catalyzing enzymes and miRNAs. (C) Mechanisms to ensure the inflammatory response does not carry on uncontrollably also involve MVs. Degranulating neutrophils also release MVs that result in decreased NF-kB signaling, as well as the release of anti-inflammatory cytokine TGF-b from macrophages. The removal of inflammatory MVs from the circulation by endothelial cells also contributes to the resolution of inflammation. Lastly, MVs containing sonic hedgehog induce NO release and proliferation of endothelial cells.

biosynthesis (Fig. 1B) [35]. Secretion of miRNA, which regulates protein expression by targeting mRNA for degradation to prevent translation, is also involved in cell recruitment to a wound. Although the mechanism of migration induction is not entirely clear, activated monocytes selectively secrete immunerelated miRNAs in MVs, specifically miR-150, which are able to induce endothelial cell migration (Fig. 1B) [36]. Further activation of the recruited cells is promoted by leukotrienes, which induce the release of NO and cytokines, such as IL-1, MCP1, and IL-6, from neutrophils and monocytes [35, 37–40]. Once neutrophils are recruited to the site of insult, they secrete MVs, as well as degranulate to eliminate any invading microbes. This degranulation is highly inflammatory and frequently causes tissue damage. The MVs from neutrophils can counter this proinflammatory response by inducing the release of TGF-b1 from macrophages (Fig. 1C) [41]. Phosphatidylserine on MVs is speculated to induce tyrosine protein kinase Mer signaling, which leads to suppression of NF-kB activation via the PI3K pathway (Fig. 1C) [41, 42]. This reduction in activation of NF-kB is required for the resolution of inflammation, and concurrently released TGF-b1 decreases proinflammatory MV release from macrophages, further contributing to the resolution of inflammation at the wound site [35].

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The immunomodulatory properties of MVs are tempered by removal from circulation by endothelial cells via endocytosis mediated by Del-1 (Fig. 1C) [43], a molecule expressed and secreted by endothelial cells, which binds to phosphatidylserine and integrins on circulating MVs and causes the vesicles to be absorbed by endothelial cells, resulting in resolution of inflammation and thrombosis [43, 44]. During the resolution of inflammation, proliferation and angiogenesis are required to repair damaged tissues at the site of insult. The sonic hedgehog protein is required for neovascularization. Activated T cells secrete sonic hedgehogcontaining MVs that induce NO release from endothelial cells, leading to optimal healing (Fig. 1C) [45, 46]. Sonic hedgehogcontaining MVs and MVs from endothelial progenitor cells can be incorporated into endothelial cells and initiate proliferation and organization of the endothelial cells into capillary-like structures [47, 48].

HOSTS AND PATHOGENS USE MVS In addition to their role in tissue repair and the resolution of inflammation, MVs are involved in the dissemination of and defense against infectious diseases. Eukaryotic cells are known to



produce MVs upon pathogen invasion, and pathogens use MVs to increase virulence and evade the host immune system.

MV secretion in viral infections MVs in viral pathogenesis and spread. Viral pathogenesis and spread are traditionally known to occur with viral replication, resulting in cell death and tissue damage. The host immune response is responsible for controlling viral replication, keeping harm to a minimum. Whereas various mechanisms of immune evasion exist, some viruses co-opt MV production to evade the host immune response and culminate in increased pathogenesis. For example, the EBV, a DNA virus belonging to the Herpesviridae family, causes various cancers. It establishes latency within these tumors, and a chief viral oncoprotein is LMP1, which is secreted in MVs of infected cells and has been found to be circulating in infected individuals with cancer attributed to EBV [49, 50]. In cells expressing LMP1, an increase in FGF2 mRNA and protein levels is observed (Fig. 2A). LMP1-expressing cells also secrete the growth factor and epidermal growth factor receptor via MVs [51, 52]. These factors are commonly overexpressed in many cancers and are critical in vasculature growth and cell proliferation. LMP1 is a major contributor to viral oncogenesis, and secretion of FGF2

is one mechanism by which LMP1 causes pathogenesis in infected and uninfected cells [51, 53]. Other viral proteins, such as negative factor (of HIV) and envelope glycoprotein E2 (of hepatitis C virus), have also been shown to be secreted via MVs, resulting in increased pathogenesis [54, 55]. Viruses use MVs additionally to facilitate viral spread. As mentioned previously, cells often shed surface proteins in MVs as a method of intercellular communication. This can, however, become a liability to the host, as in the case of chemokine receptor CCR5 transfer (Fig. 2B). This receptor is normally expressed on macrophages and T cells and is the primary coreceptor used by HIV to infect macrophages [56]. However, HIV appears to infect cell types that do not express CCR5 or CXCR4. An observation by Mack et al. [57] elucidated that this phenomenon occurred by transfer of CCR5 between cells via MVs. In a series of elegant experiments, it was demonstrated that PBMCs released CCR5 in MVs and that cells that were deficient in CCR5 could receive these MVs and subsequently express CCR5 on their surface. Consequently, HIV could productively infect these previously CCR5-negative cells with HIV. Likewise, the coreceptor CXCR4 used by T cell tropic HIV was confirmed to transfer via MVs from megakaryocytes and platelets

Figure 2. MVs in viral infections. In the context of a viral infection, MVs can function to protect the host (C–F) or facilitate viral spread (A and B) and immune evasion (G and H). (A) MVs shed from infected cells can induce growth and replication in recipient cells. This can lead to cell proliferation and oncogenesis. (B) MVs contain cellular receptors and can lead to infection of previously nonsusceptible cells. (C) MVs shed from infected cells can directly present antigen to effector cells. (D) MVs shed from infected cells are also absorbed by APCs, which can then present this antigen to effector cells. (E) MVs shed from infected cells may also initiate immune signaling in non-APCs. (F) MVs contain viral receptors and can neutralize the viruses via binding directly to the particle. (G) MVs contain miRNAs that modify protein expression in the recipient cell. (H) MVs are shed from infected cells that contain proteins involved in the host immune response, allowing for viral replication. Light blue vesicles, SVs (left); dark blue vesicles, exosomes (right).

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to CXCR4-negative cells [58]. This transfer rendered cells susceptible to the T cell tropic virus [58]. MVs in host defense toward viral infections. One of the numerous ways in which cells respond to viral infections is antigen presentation. Peptide presentation by APCs via MHC results in activation of effector immune cells, such as T cells. It was shown by Raposo et al. [59] that B cells latently infected with EBV could secrete peptide-loaded MHC II contained in MVs, which were capable of activating T cells (Fig. 2C). This was the first study to show that these complexes were secreted on vesicles from APCs and also had the ability to stimulate an immune response. Importantly, it is also a way of efficiently amplifying the adaptive immune response, as DCs do not need to internalize and reprocess antigens on MVs to present them [60]. APCs, such as DCs and the aforementioned B cells, secrete MVs that contain peptide-loaded complexes, which are then absorbed by other APCs, allowing those uninfected cells to present the antigen to effector cells (Fig. 2D) [59, 61–63]. The MVs also fuse with nonAPCs that are not able to present antigen but can initiate innate immune signaling cascades in response to other MV content, such as cytokines and receptors (Fig. 2E) [64]. Host defense mechanisms, by use of MVs before encountering pathogens, have also evolved. Human tracheobronchial epithelial cells were examined for vesicle release by Kesimer et al. [65]. It was discovered that the cells did indeed release MVs, and those MVs had high-surface expression of sialic acid, the cellular receptor used by the influenza virus. Furthermore, the MVs were able to blunt infection of the influenza virus when the 2 were coincubated before infection, indicating that the MVs competitively bound viral particles and may act as a decoy (Fig. 2F) [65]. In another example of MV secretion for host defense, cytidine deaminase apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G, a cellular nucleic acid editing protein with antiretroviral function, was found to be secreted in MVs and also to inhibit HIV replication in recipient cells [66]. The catalytic editing protein is speculated to inhibit extensive viral replication by destabilizing or preventing nuclear transport of the viral DNA [66]. Host defense via miRNA-containing MVs. A rapid expansion in the area of miRNA research has occurred since the discovery of the short, noncoding RNA in the early 1990s [67]. These small molecules have been shown to be involved in every aspect of biology, from development to immune regulation. It now has been shown that miRNAs are also secreted in MVs, along with the cellular machinery required to process them (e.g., processing enzymes) [8, 68]. In response to infection, cells produce miRNAs to regulate antiviral and proinflammatory protein expression, and although there is no definitive evidence that MV-encased miRNAs influence recipient cells toward an antiviral state, several lines of evidence suggest that this is possible. Antigen-specific T cells were shown to form an immunologic synapse with APCs, through which, miRNAs were transferred unidirectionally via exosomes [69]. Transfer of miRNAs from T cells to APCs was antigen driven and dependent on the formation of the immunologic synapse [69]. This shows a targeted transfer via MVs when T cells are activated. Another recent study examined the miRNA content of MVs from mature and immature DCs and found that the miRNA composition varied depending on the

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state of maturity [70]. Stimulated DCs contained a different repertoire of miRNAs, which were functionally active when transferred via MVs to recipient DCs (Fig. 2G) [70]. Both studies indicate that cells sort and selectively secrete miRNAs in response to stimuli, such as infection, although the mechanism of sorting is unknown. Hijacking MVs for immune evasion. As with other aspects of host defense, viruses have evolved to use the secretion pathway to their benefit. DNA viruses in the Herpesviridae family have been well studied in the area of immune evasion, and recent studies have shown that they have acquired mechanisms to manipulate the cell’s secretory pathways. The Alphaherpesvirus HSV-1 is a ubiquitous virus that usually infects initially during childhood. It remains latent in neurons, reactivating at irregular intervals, as a result of stimuli, such as stress or immune suppression. Hosts are unable to clear the infection fully, as a result of the many evasion strategies that the virus has evolved. One of these evasion mechanisms is the HSV-1 gB binding to HLA-DR, thus inhibiting its binding to the invariant chain and ultimately impeding peptide binding and presentation [71]. This interaction also prevents HLA-DR from being expressed at the surface. MVBs are formed during the process of peptide loading, where sorting takes place. The binding of gB to HLA-DR was found to occur in MVBs as both proteins colocalized with microvesicular markers [72]. This same study went on to show that MVs secreted from gB-expressing cells contained gB and HLA-DR (Fig. 2H). The viral and host proteins remain bound in the MV as they were in the cell. As HLA-DR is prevented from binding peptides and expelled from the cell without being expressed on the plasma membrane, this provides a tantalizing suggestion that HSV-1 may be able to evade the host immune defense by redirecting key MHC proteins to the exosomal pathway. Another mechanism of immune evasion is secretion of miRNA in MVs. As discussed previously, host cells can use this pathway to signal to neighboring cells via miRNA. Virally infected cells, including EBV-infected cells, have also been shown to secrete miRNA encoded by the infecting virus. B Cells infected with EBV secrete virally encoded miRNAs that are able to prevent expression of proapoptotic viral protein LMP1 and CXCL11, a T cell chemoattractant, in recipient cells (Fig. 2G) [73]. Expression of LMP1- and CXCL11-specific miRNAs promotes the maintenance of latency and consequential immune evasion [74, 75]. The phenomenon of EBV miRNA transfer was also seen in patients latently infected with EBV, where viral miRNA was detected in uninfected T cells and monocytes [73]. Although downstream effects, such as suppression of apoptosis, were not examined, detection of the viral miRNA in cells that did not express viral DNA indicates that circulating MVs from EBV-infected cells transfer miRNAs in vivo as well as in vitro [73].

MV secretion in bacterial infections Bacterial production of OMVs. As outlined in Table 1, bacteria produce and secrete MVs with characteristics distinct from eukaryotic vesicles. Whereas Gram-negative and -positive bacteria produce vesicles, the majority of prokaryotic vesicle research has been in Gram-negative bacteria, as the discovery of Gram-positive vesicles occurred recently in 2009 [83]. As previously mentioned,



all Gram-negative bacteria examined have been found to secrete OMVs, whereas the study of Gram-positive MVs is in its infancy. This disparity is mainly a result of structural differences between the bacteria. Gram-negative bacteria possess an outer membrane from which many groups have confirmed these vesicles originate, whereas Gram-positive bacteria do not. It was assumed that the same vesicles could not be secreted from Gram-positive organisms because of the thick cell wall. Although there are now several studies supporting the secretion of OMV from Grampositive species [83, 84], most of what will be discussed is research performed on Gram-negative species. OMVs in bacterial pathology. What was first proposed in the 1960s has now been confirmed: OMVs are mechanisms for toxin secretion by many pathogenic bacteria. Toxins can be absorbed by host cells and have a variety of detrimental effects. One wellstudied opportunistic respiratory pathogen is Pseudomonas aeruginosa. It is a common nosocomial pathogen found to colonize hospital equipment, such as catheters and ventilators, and causes pneumonia in immunocompromised patients. P. aeruginosa produces a number of toxins that have been detected in OMVs released from the bacteria, including LPS, which is commonly found in OMVs secreted from Gram-negative bacteria [85, 86]. The toxin-containing OMVs were able to fuse with airway epithelial cells and travel through a thick mucus layer, ultimately causing epithelial cell death (Fig. 3A) [86]. This observation helps explain the well-documented phenomena of lung damage that occurs in the absence of direct cell-bacteria contact. Other examples of toxin-containing OMVs can be seen in Helicobacter pylori, which release a vacuolating toxin (vacuolating cytotoxin A) [87]. This toxin is delivered to adjacent and distal gastric cells via OMVs, subsequently inducing apoptosis, and could partially explain the pathology of H. pylori-colonized patients suffering from gastric ulcers [87, 88]. MVs and OMVs in host defense. Intracellular bacteria, such as mycobacteria, have been known to grow in phagosomes that are in close contact with the secretory pathway. Knowing this, it is not surprising that macrophages infected with these bacteria, such as Mycobacterium bovis, Mycobacterium tuberculosis, and Salmonella serotype Typhimurium, secrete MVs that contain bacterial proteins. These pathogen-associated molecular patterns containing MVs are then able to stimulate a proinflammatory response in recipient cells via TLR signaling (Fig. 3B) [89, 90]. Recipient cells can, in turn, secrete MVs that express MHC II and costimulatory molecules that initiate a proinflammatory response in epithelial cells [64, 91]. When incubated with macrophages, MVs from infected cells were able to stimulate TNF-a, RANTES, and iNOS production [89, 92]. Another species of opportunistic mycobacteria, Mycobacterium avium, which is commonly found in patients with HIV, has also been shown to induce secretion of proinflammatory MVs from macrophages [90]. OMVs from bacteria themselves can also elicit a potent immune response. Most Gramnegative bacteria have been found to secrete LPS contained within OMVs, which then can stimulate an innate immune response in the host. S. Typhimurium has been shown to produce OMVs that have the ability not only to activate the innate immune response by inducing DC maturation but also to generate an adaptive immune response [92]. OMVs from S. Typhimurium were injected into mice, and an antigen-specific IgG response 6



Figure 3. MVs in bacterial infections. Vesicles shed from bacteria can cause pathology (A and C) and aid in development of antibiotic resistance (D). Bacterially infected cells also secrete MVs in host defense (B). (A) OMVs containing bacterial toxins induce apoptosis of recipient cells. (B) MVs from bacterially infected cells can initiate immune signaling in recipient cells. (C) OMVs containing bacterial toxins that cause degradation of cellular proteins in recipient cells. (D) OMVs contain enzymes that target antibiotics, such as b-lactam, rendering antibiotics useless before reaching the bacteria itself. Green vesicles, OMVs (upper); light blue vesicles, SVs (lower); dark blue vesicles, exosomes (lower).

was seen in the sera along with Salmonella-specific CD4+IFN-g+ T cells [92]. Furthermore, this priming protected mice from a subsequent S. Typhimurium challenge [92]. Evasion tactics by use of OMVs. As with any other pathogen, bacteria have evolved to possess evasion tactics to confer resistance to antibiotics, further colonization, or even to eliminate other competing species of bacteria. Most of these strategies involve toxins and proteins secreted from the bacteria, and they are commonly contained within OMVs. Host-cell processes are impacted by these bacterial toxins to the benefit of the bacteria. The aforementioned P. aeruginosa is a significant pathogen in immunocompromised patients, and it is also the cause of significant morbidity in patients with cystic fibrosis. The disease is caused by genetic mutations in the gene encoding the

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chloride ion channel CFTR and results in lowered expression of the transmembrane protein, reduced chloride secretion, and a build-up of mucus in the lung. P. aeruginosa expresses and secretes a virulence factor Cif in OMVs that down-regulates expression of CFTR further by targeting it for degradation in the lysosome (Fig. 3C) [86, 93]. Cif stabilizes the interaction of ubiquitin-specific peptidase 10, the enzyme that deubiquitinates CFTR, with its inhibitor, resulting in an increase in ubiquitinated CFTR, which is subsequently degraded [93]. The resulting increase in mucus build-up further reduces the patient’s ability to clear respiratory pathogens by mucociliary clearance and allows P. aeruginosa to persist in the lungs. Antibiotic resistance rates are rising in many bacterial pathogens, and OMVs play a role in resistance to commonly used antibiotics. For example, antibiotics frequently used to treat P. aeruginosa are b-lactam antibiotics, but resistance is quickly developed as a result of production of b-lactamase by the bacteria. The enzyme is usually periplasmic, where it can cleave b-lactam before it is able to reach the bacterial cytoplasmic membrane. Ciofu et al. [94] were the first to show that b-lactamase is also secreted from P. aeruginosa in OMVs (Fig. 3D). The protein responsible for b-lactam antibiotic resistance was detected in secreted vesicles and explains why there are such high levels of b-lactamase in the sputum of cystic fibrosis patients on these antimicrobial treatments [94]. Furthermore, it was shown that another pathogen that colonizes the nasopharyngeal tract, Moraxella catarrhalis, also secretes b-lactamase containing OMVs [94]. These OMVs were able to fully hydrolyze amoxicillin, rendering it ineffective against susceptible strains, and confer resistance to b-lactam-susceptible species Streptococcus pneumonia and Haemophilus influenza, both commonly coisolated with M. catarrhalis [95]. All isolates of M. catarrhalis in this study secreted OMVs containing b-lactamase and imply that if colonized, patients with multiple infections could quickly become resistant to b-lactam antibiotic treatments.

CONCLUSIONS The discovery of MVs has explained the mechanisms behind many phenomena; however, many questions still remain. For example, the mechanism by which their content is determined is not described definitively. A frontrunner in the theory is that localization of proteins to lipid rafts is necessary to be secreted, but it seems that lipid-raft localization is not the sole determinant for excretion. Another looming question is that of targeting. Are some or all MVs destined for a specific cell type? In certain cases, close cell proximity is necessary for transfer, but it appears that MVs circulate in the body and can travel long distances to reach their target. More research is needed on these mysterious cell communicators to answer these physiologically important questions. It is increasingly clear that as we learn more of these small vesicles once thought to be merely cell debris, laboratory studies on MVs must be approached with caution and clarity. For example, one must be certain to differentiate between apoptotic blebs and secreted MVs, as these are 2 very different types of vesicles in origin and downstream effects. One possible method would be by use of dyes, such as propidium iodide and Annexin V, to check the state of the source cells, which could aid in confirming that the source is

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nonapoptotic. The inhibition of apoptosis with a caspase inhibitor before MV isolation would also build confidence that effects are from MVs and not apoptotic bodies. These actions would not only help researchers in the field to replicate phenomena more accurately but also to be sure of the MV species to which they are attributing these phenomena. For the researchers whose focus may not be MVs, it may be worthwhile to consider the role of MVs in everyday experiments. As our scientific forefathers have discovered time and again, unexpected observances are often phenomena. We now know that the debris and particles other than cells typically seen during flow cytometry are most likely MVs. An amplified response seen in in vitro cell culture could be attributed to secreted MVs. Your next unexpected result might very well be a result of this tiny contributor.

ACKNOWLEDGMENTS Work in the D.M.E.B. lab is funded, in part, by the Canadian Institutes of Health Research and Natural Sciences and Engineering Research Council of Canada. Research in the D.M.E.B. and B.D.L. labs is supported by the M. G. DeGroote Institute for Infectious Disease Research and the McMaster Immunology Research Centre. DISCLOSURES

The authors declare no competing financial interests. REFERENCES

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KEY WORDS: intracellular communication host-pathogen interactions wound healing ectosome exosome shedding vesicle •

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Immunity to infection.

Heparan sulfate: a ubiquitous glycosaminoglycan with multiple roles in immunity.

Quantitation of cell-mediated immunity: responses to dinitrochlorobenzene and ubiquitous antigens.

Guidelines to Contributors.

Instructions to Contributors.

Instructions to Contributors.

Instructions to Contributors.

Guidelines to Contributors.

Suppression of immunity to Mycobacterium lepraemurium infection.

Protective immunity to chronic bacterial infection.

Innate immunity to influenza virus infection.

Genetic and epigenetic contributors to FSHD.

Cell-mediated immunity and its role in resistance to infection.

Acquired and natural immunity to gonococcal infection in chimpanzees.

Preface to the special issue: zebrafish immunity and infection models.

Microbial programming of systemic innate immunity and resistance to infection.

Environmental contributors to autism: the pediatrician's role.

Haemophilus parasuis: infection, immunity and enrofloxacin.

Statistics in Infection and immunity revisited.

Mechanisms of immunity in typhus infection: adoptive transfer of immunity to Rickettsia mooseri.

Dietary contributors to hypertension in adults reviewed.

Fathers: forgotten contributors to child development.

Organic contributors to the intermetamorphosis syndrome.

Occupational therapists as contributors to health promotion.