Extracellular vesicles and asthma: A review of the literature

Asthma is a chronic, recurrent and incurable allergy‐related respiratory disease characterized by inflammation, bronchial hyperresponsiveness and narrowing of the airways. Extracellular vesicles (EVs) are a universal feature of cellular function and can be detected in different bodily fluids. Recent evidence has shown the possibility of using EVs in understanding the pathogenesis of asthma, including their potential as diagnostic and therapeutic tools. Studies have reported that EVs released from key cells involved in asthma can induce priming and activation of other asthma‐associated cells. A literature review was conducted on all current research regarding the role and function of EVs in the pathogenesis of asthma via the PRISMA statement method. An electronic search was performed using EMBASE and PubMed through to November 2018. The EMBASE search returned 76 papers, while the PubMed search returned 211 papers. Following duplicate removal, titles and abstracts were screened for eligibility with a total of 34 studies included in the final qualitative analysis. The review found evidence of association between the presence of EVs and physiological changes characteristic of asthma, suggesting that EVs are involved in the pathogenesis, with the weight of evidence presently favouring deleterious effects of EVs in asthma. Numerous studies highlighted differences in exosomal contents between EVs of healthy and asthmatic individuals, which could be employed as potential diagnostic markers. In some circumstances, EVs were also found to be suppressive to disease, but more often promote inflammation and airway remodelling. In conclusion, EVs hold immense potential in understanding the pathophysiology of asthma, and as diagnostic and therapeutic markers. While more research is needed for definitive conclusions and their application in medical practice, the literature presented in this review should encourage further research and discovery within the field of EVs and asthma.

may occur. Interaction between diverse types of cells through mediators often results in airway inflammation and obstruction, as summarized in Figure 1. [5][6][7][8] Extracellular vesicles (EVs) are a universal feature of cellular function and can be detected in culture supernatants of different bodily fluids such as serum, bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NLF). 9 Due to these characteristics, there has been an increased interest in EVs and their potential applications in understanding the underlying mechanisms of various lung diseases, their use as biomarkers and therapeutic tools in lung inflammation. 10 EVs are classified into three classes based on their biogenesis, secretory components and size: exosomes, microvesicles (ectosomes) and apoptotic bodies. 11 Exosomes are 50-150 nm EVs of endosomal origin. They contain enriched amounts of certain surface markers including tetraspanins, heat shock proteins and MHC classes I and II. These components can be transferred to target cells by either direct membrane fusion, endocytosis, phagocytosis or ligand interaction. 12,13 Microvesicles (MVs) are larger than exosomes (100-2000 nm) and are derived from the plasma membrane of cells through direct outward budding. They contain substantial amounts of phosphatidylserine and membrane components as do their parent cells. Both exosomes and MVs contain elements, which allow them to act as intercellular facilitators and release relevant signalling molecules. 10,14 Apoptotic bodies are released from cells that undergo apoptosis and are usually 1-4 μm in diameter. They may contain DNA fragments, non-coding RNAs and cell organelles. 15 Recent evidence has shown the possibility of using EVs in understanding the pathogenesis of asthma, including their potential as diagnostic and therapeutic tools. Methods such as ultracentrifugation and ExoQuick precipitation allow EVs to be isolated and studied in detail.
Studies have reported that EVs released from key cells involved in asthma can induce priming and activation of other asthma-associated cells. The production of these EVs can be regulated by pro-inflammatory and oxidative stress stimuli in vitro. [16][17][18] For example, exosomes released from DCs containing costimulatory molecules and MHC class II can activate immune functions of Th2 cells within the lungs. 19 A multitude of research studies have investigated different pharmacological targets in asthma to better understand the pathophysiology of the disease and find novel treatments. In this respect, EVs have great potential, but there is currently insufficient research. In other respiratory diseases, EVs have already shown their potential. For example, microvesicles released from mesenchymal stem cells (MSCs) have been found to be cytoprotective in pulmonary fibrosis, a disease which is currently incurable. 20 DC-derived exosome-based cell-free vaccines have shown success in eradicating established murine tumours. 21 Much ongoing research exploring the potential of exosomes in chronic obstructive pulmonary disease (COPD), lung cancer, cystic fibrosis, primary ciliary dyskinesia, along with many others, is being conducted. 22 This article reviews all available research on EVs in the pathogenesis of asthma to identify existing knowledge and promote further research within the field. Highlighting hypotheses and noteworthy results from current literature also underlines the limitations of past studies, for future improvements. The specific research question we address is whether there is currently sufficient evidence to support pro-inflammatory, airway remodelling and/or protectives roles of cell-derived EVs in asthma.

| Sources and searches
For this review, the PRISMA statement method was implemented.
An electronic search was performed using EMBASE and PubMed (results up to November 2018).

| Study selection
The aim of this review was to combine and interpret all current research on the role and functions of EVs of known cellular origins in the pathogenesis of asthma. Only primary research literature has been included. Research that was either unpublished or published in non-peer-reviewed forms (including abstracts and conference proceedings) was not included. Studies concerning exosomes derived from microorganisms and their effects in asthma were also excluded.

| Results of PRISMA statement evidence search and selection
The EMBASE search returned 76 papers, while the PubMed search returned 211 papers. Search results were imported using the reference manager, ENDNOTE, to find and exclude duplicates. After duplicates were removed (reducing the number to 260), titles and abstracts were then screened for relevance to the research question: this led to further exclusions due to lack of relevance (eg wrong type of "microparticles") or because publications were reviews rather than primary research (records excluded n = 197).
The remaining 63 studies were eligible for a full-text review. A further 29 studies were excluded using inclusion and exclusion criteria (Table 1)

| Extracellular vesicles of known cellular origin
Twenty-one studies found that EVs of known cellular origin are associated with asthma and allergic inflammation. Of these, four F I G U R E 1 Mechanisms of Airway Inflammation in Asthma. In the sensitization phase, DCs present antigen to T cells in the lymph nodes causing differentiation of naïve T cells. The Th1 response leads to cell-mediated immunity and neutrophilic inflammation. Th2 cytokines cause class switching of B cells to produce IgE (mainly through IL-4), and recruitment of immune cells including eosinophil (mainly through IL-5). In the early asthmatic response, allergen binding to IgE receptors on mast cells causing degranulation and release of histamine, proteases, tumour necrosis factor (TNF), lipid mediators and other proteins. This leads to immediate hypersensitivity and bronchoconstriction. In the late asthmatic response, mast cells release chemokines and cytokines, which recruit eosinophil, basophils (not shown) and T helper cells to the local mucosa. Both mast cells and Th2 cytokines lead to eosinophilia. Eosinophils degranulate to release eosinophil granule proteins. These proteins cause bronchoconstriction along with damage towards the epithelial layer. Ongoing inflammatory responses in the airway can lead to smooth muscle dysfunction and airway remodelling  Figure 4).

| Mast cell-derived extracellular vesicles
Mast cells release histamine, prostaglandins, leukotrienes and other inflammatory mediators, which are capable of inducing an asthma attack (Table 2). 23

| Eosinophil-derived extracellular vesicles
Eosinophils are often found in severe types of asthma and are amongst the main effector cells in the disease (Table 2). Elevated levels of eosinophils are found to cause airway inflammation and breathlessness. 27,28 Four trials suggested that exosomes from eosinophils are associated with asthma and the lung inflammatory response (Table 2). Mazzeo et al 29 investigated exosome secretion by eosinophils. Exosomes were purified from eosinophils in the peripheral blood of asthmatic and healthy subjects. Results confirmed the secretion of exosomes by eosinophils and found that interferon-gamma (IFN-γ) stimulation led to an increase in the production of exosomes. Furthermore, they demonstrated that the production of exosomes was augmented in asthmatic patients. 29 Akuthota et al 30  Canas et al 31 reported that the amount of exosome production was greater from eosinophils of asthmatic patients compared to healthy individuals. The rate of apoptosis of eosinophils of asthmatic patients was also reduced compared to healthy participants.
The exosomes increased eosinophil production of ROS and NO, and and POSTN (periostin) genes, which represent an asthma-specific mRNA signature. 33 Hyperplasia and hypertrophy of BMSC is thought to be part of the pathophysiology of the process. Data from the study showed significantly increased proliferation of BMSC when co-cultured with exosomes from eosinophils of asthmatic patients, as well as increased gene expression of CCR3 and VEGF-A, which are associated with angiogenesis and fibrotic processes. 32 In summary, the study shows significant involvement of eosinophil-derived exosomes in the pathophysiology and disease progress of asthma.

| Neutrophil-derived extracellular vesicles
Although asthma has been classically associated with eosinophils, newer research has found that neutrophils may also play a role in the inflammation and airway remodelling of asthmatics, especially in severe cases (Table 2). 34

| B cell-derived extracellular vesicles
B cells play a crucial role in the sensitization and adaptive immune response of asthma (Table 3). 38 and TNF-α). 43 The mechanism by which B cell-derived exosomes activate T cells is still under debate, but some studies suggest that exosomes can stimulate T cells directly, 42,44 whereas others demonstrate that exosomes need the presence of antigen-presenting cells to exert their effects. 45,46

| T cell-derived extracellular vesicles
Th2 cell-derived inflammation, leading to airway responsiveness and tissue remodelling, is one way asthma can be characterized (

| Dendritic cell-derived extracellular vesicles
Dendritic cells (DCs) play a central role in sensing the presence of foreign antigens and infectious agents and presenting them to T cells (Table 3). DCs also regulate the activation of allergic pathways in response to potential environmental allergens. Significant increases in the numbers of airway DCs have been reported after exposure to allergen and can lead to an inflammatory response. 53

| Myeloid-derived regulatory cell extracellular vesicles
Free radical-generating MDRCs are known to regulate activities of T cells and airway responses in asthma (Table 3). 55

| Platelet-derived extracellular vesicles
Platelet microparticles (PMPs) make up the largest fraction of circulating EVs and are elevated in many systemic diseases such as rheumatoid arthritis, cancer, diabetes and acute coronary syndrome (

| Epithelial cell-derived extracellular vesicles
Airway epithelial cells play a pivotal role in the pathogenesis of asthma as a primary airway defence against exposure to inflammatory stimuli and antigens ( Table 4). Activation of epithelial Toll-like receptors provides an important link between innate immunity and allergic diseases. 62 Epithelial cells can also promote inflammation by directing DCs to drive a Th2 response. 63

| Fibroblast-derived extracellular vesicles
By regulating the functions of bronchial epithelial cells, bronchial fibroblasts play a key role in the structural changes, which occur in

| Mesenchymal stem cell-and mesenchymal cell-derived extracellular vesicles
Mesenchymal stem cells are pluripotent stromal cells, which differentiate into a variety of mesenchymal cell types including osteocytes, myocytes and adipocytes (Table 4)

| Bronchoalveolar lavage fluid-derived extracellular vesicles
Eight studies have investigated the role of exosomes derived from BALF in the pathogenesis of asthma (Table 5) A subsequent study by the same authors 80 investigated whether such exosomes generated in response to Ole e 1 could also prevent the sensitization to other unrelated allergens, such as Bet v 1 from birch pollen. Exosomes were isolated from BALF of Ole e 1 tolerized mice using the same methods as in the previous study. The results suggest that pre-treatment with tolerized exosomes specific to Ole e 1 could also block allergic responses to a second unrelated allergen such as Bet v 1.

Gon et al 81 analysed variations in the production of airway
EVs and their miRNA content in a house dust mite (HDM) allergen-exposed murine asthma model. Airway EVs from BALF of mice were isolated using the ExoQuick Exosome Precipitation Solution.
Compared to the usual technique of ultracentrifugation, this provides a higher extraction efficiency but has the risk of protein contamination when used to isolate exosomes in cell culture media. 82 The results showed that the amount of EVs observed increased by

| Serum-derived extracellular vesicles
Two studies have reported that exosomes derived from serum could potentially be involved in the pathogenesis of asthma (Table 6).

| Nasal lavage fluid-derived extracellular vesicles
Lässer et al 86  Determine whether miRNAs contained in EBCderived exosomes could be an ideal substrate for potential biomarkers in vitro The secretion and contents of EBC-derived exosomes are highly regulated and could reflect ongoing biological processes and chronic rhinosinusitis ( Table 6). Serum-associated proteins and mucins were increased in the exosomes from individuals with respiratory diseases compared to healthy controls, whereas proteins with antimicrobial functions and barrier-related proteins showed decreased expression.

| Exhaled breath condensate-derived extracellular vesicles
Sinha et al 87 demonstrated how miRNAs could be reliably detected in exhaled breath condensate (EBC) using quantitative PCR analysis (Table 6). RNA was extracted from vacuum-dried and concentrated EBC samples of asthmatic and healthy subjects. It was found that miRNAs present in EBC are mainly associated with exosomes.
The EBC miRNA profile of asthmatic patients differed from that of healthy controls, with some of the identified miRNAs known to be associated with asthma, allergy and inflammatory pathways.

| Main findings
The investigation of the biological role of EVs in lung disease has become a rapidly progressing field. The role of EVs as intercellular messengers and the possibility that they could serve as novel diagnostic or pharmacological targets has made this an exciting field. The aim of this review was to combine and interpret all current research on the role and functions of EVs in the pathogenesis of asthma and, in particular, assess the weight of evidence for specific roles of EVs in asthma. Data from the studies included in this review provide evidence that EVs, including exosomes and MVs, may play important roles in the pathogenesis of asthma and airway inflammation.

| Findings for extracellular vesicles of known cellular origins
One objective of the review was to identify all current research on

| The therapeutic potential of extracellular vesicles
An interesting aspect of exosomes is their potential in inducing tol-

| The diagnostic potential of extracellular vesicles
Whilst many studies demonstrate measurable differences between the characteristics of EVs in healthy and asthmatic patients, the difficulty of using EVs as diagnostic tools is justifying the use of invasive procedures required to obtain the EVs. Currently, the diagnosis of asthma is clinically based and does not require invasive procedures such as bronchoalveolar lavage 77 or blood tests 86 to confirm the diagnosis. However, novel non-invasive methods, such as using exhaled breath condensate to obtain EVs, 87 may be useful in helping to classify the severity of the disease and predict the prognosis. Clearly, evaluating the potential value of EVs as biomarkers of asthma requires studies involving a much more detailed comparison of EVs with other biomarkers that are already wellcharacterized and available. In addition, studies are required to investigate the relationship between particular types of EVs and different phenotypes and endotypes of asthma that have been characterized.

| Limitations and future research
Heterogeneity within the research reviewed here should be considered. The studies adopted various experimental designs from in vitro studies of human cells, in vitro studies of murine cells, in vitro studies of equine cells to in vivo studies in mice. Differences in development, gene regulation and expression, genomics and epigenetics between each species should be considered before extrapolating the results to humans. 88 Furthermore, the numbers of subjects (patients and controls) are also critical in assessing the confidence with which conclusions can be made: most of the studies discussed in this review employed relatively low numbers of patients or healthy controls (≤20), with some having only three or four subjects. The highest number was in a study employing 58 asthmatic subjects and 16 healthy volunteers to study exosomes derived from eosinophils. 31 Thus, more studies with larger numbers of subjects are required in order to consolidate confidence in the findings reported. The heterogeneity of patients in terms of the representation of different phenotypes and endotypes of asthma within the subject populations should also be considered.
Due to the relatively small number of currently available studies in each area of research, the results and findings presented must be interpreted with caution. Thus, further studies are required to consolidate the evidence that is currently available. Every area presented in this review potentially needs more research to be conducted; however, some areas hold more promise than others. For example, additional research on the differences in exosomal contents or levels between asthmatic and healthy subjects could help to develop a novel, non-invasive diagnostic tool for asthma, particularly by investigating EVs in exhaled breath. 87 The role of EVs in preventing allergic sensitization is also a fascinating area with the potential for developing preventative or prophylactic medicines; however, it is likely to be a significant time before such studies can move from animal models to patients. Because myriads of cells within the body can produce exosomes, each containing distinct constituents, the possibility for further research is considerable.
A further area for research is likely to be the effects of allergen desensitization interventions on the nature and constituents of EVs as this could provide further insights into the relationship between EVs and asthmatic status. TA B L E 7 The role of each EV separated by cell origin in asthma Overall, the studies conducted so far on EVs in relation to asthma indicate that they are significant factors in the disease, but much further research is required to consolidate and expand on these findings.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed in this study.