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Intercellular Communication by Extracellular Vesicles and Their MicroRNAs in Asthma

      Abstract

      Purpose

      Extracellular vesicles (EVs) such as exosomes and microvesicles are phospholipid bilayer–enclosed vesicles that are recognized as novel tools for intercellular communications and as biomarkers for several diseases. They contain various DNAs, proteins, mRNAs, and microRNAs (miRNAs) that have potential diagnostic and therapeutic purposes. Their biological roles have attracted significant interest in the pulmonary field because their vesicle composition and miRNA content have the ability to transfer biological information to recipient cells and play an important role in pulmonary inflammatory and allergic diseases. Asthma is a chronic inflammatory disease of the airways, and it is characterized by variable and recurring symptoms and reversible airflow obstruction. The purpose of this review was to discuss the function of EVs and their miRNAs in asthma, with a focus on the biological properties and biogenesis of EVs, their pathophysiologic roles, and their potential use as biomarkers and therapies for asthma.

      Methods

      We review the findings from several articles on EVs and their miRNAs in asthma and provide illustrative references.

      Findings

      A few studies have reported on the biological function of bronchoalveolar lavage fluid–derived EVs in asthmatic progression. In the lungs, EVs might regulate airway inflammation and allergic reactions through their paracrine effects. Furthermore, circulating miRNAs have been found to be associated with EVs.

      Implication

      EV-mediated miRNAs can be used as biomarkers in asthma.

      Key words

      Introduction

      Cell-to-cell communication is one of the key mechanisms of lung disease biology. In multicellular organisms, intercellular communication is believed to involve the secretion of soluble factors such as cytokines and chemokines. Over the past decade, extracellular vesicles (EVs) have become the next focus of intensive scientific research as novel mediators of intercellular communication. In this review, we use the term EVs, in accordance with a recommendation of the International Society for Extracellular Vesicles, as an umbrella term for all types of vesicles present in the extracellular space, including exosomes and microvesicles.
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      As we wait: coping with an imperfect nomenclature for extracellular vesicles.
      Although they have been known to exist for several decades, membrane vesicles have long been thought of as cell debris, signs of cell death, and/or structures that are very specific to a unique organ. However, recent studies have suggested that EVs might be important tools as diagnostic markers of, and as new potential therapeutic targets for the clinical treatment of, various lung diseases.
      • Eissa N.T.
      The exosome in lung diseases: message in a bottle.
      EVs, especially exosomes, are small membrane vesicles released into the extracellular space after the fusion of multivesicular endosomes with the cell membrane.
      • Thery C.
      Exosomes: secreted vesicles and intercellular communications.
      Exosomes contain enriched amounts of some specific markers, especially those of endosomal origin, including CD9, CD63, CD81, heat shock 70-kDa protein 4, and major histocompatibility complex class II (Figure 1).
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      Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes.
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      Comparative marker analysis of extracellular vesicles in different human cancer types.
      Healthy cells shed microvesicles from the plasma membrane.
      • Raposo G.
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      Extracellular vesicles: exosomes, microvesicles, and friends.
      Although EVs likely comprise both exosomes and microvesicles, it is difficult to fully discriminate between these 2 types of EV. Their difference is based on their diameters: Exosomes are in the range of 10 to 100 nm, and microvesicles are in the range of 100 to 1000 nm.
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      The therapeutic potential of mesenchymal stem cell-derived extracellular vesicles.
      Figure thumbnail gr1
      Figure 1The molecular components and biological significance of extracellular vesicles (EVs). EVs contain various proteins on the surface membrane and inside the luminal space. Moreover, EVs package DNA, mRNA, and microRNAs. EVs are recognized as a novel tool of cell-to-cell communication and as biomarkers for several diseases.
      A wide variety of cell types have been shown to release EVs, including immune cells, epithelial cells, and tumor cells. EVs are reportedly released from both immune and structural cells in the lungs, and they have recently been reported to play a role in allergies and asthma.
      • Admyre C.
      • Telemo E.
      • Almqvist N.
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      Exosomes - nanovesicles with possible roles in allergic inflammation.
      • Torregrosa Paredes P.
      • Esser J.
      • Admyre C.
      • et al.
      Bronchoalveolar lavage fluid exosomes contribute to cytokine and leukotriene production in allergic asthma.
      EVs have been isolated and characterized from different bodily fluids, such as plasma,
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      • Raposo G.
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      Exosomal-like vesicles are present in human blood plasma.
      urine,
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      Proteomic analysis of podocyte exosome-enriched fraction from normal human urine.
      and bronchoalveolar lavage fluid (BALF).
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      • Grunewald J.
      • Eklund A.
      • Gabrielsson S.
      Proinflammatory exosomes in bronchoalveolar lavage fluid of patients with sarcoidosis.
      Previous studies have suggested that EVs might be involved in a broad range of biological processes, including immune system regulation, inflammation, and tumor development.
      • Thery C.
      Exosomes: secreted vesicles and intercellular communications.
      It has been suggested that EVs carry communications between cells, allowing for cells to promote biological functions at distant sites.
      • Bang C.
      • Thum T.
      Exosomes: new players in cell-cell communication.
      A breakthrough in EV research was the finding that their nucleic acid contents, such as mitochondrial DNA, messenger RNA (mRNA), and microRNA (miRNA), can be transported to recipient cells. In the EVs, these contents are protected from enzymatic degradation. In 2006, it was reported that EVs contain and transfer mRNAs to recipient cells, where these cargo mRNAs are translated into proteins.
      • Ratajczak J.
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      • et al.
      Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery.
      In 2007, Valadi et al
      • Valadi H.
      • Ekstrom K.
      • Bossios A.
      • et al.
      Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.
      reported that EVs are enriched in miRNAs called exosomal shuttle RNAs. miRNAs are emerging as novel therapeutic targets and diagnostic biomarkers for an array of disorders, including various lung diseases.
      • Greene C.M.
      • Gaughan K.P.
      microRNAs in asthma: potential therapeutic targets.
      EVs have garnered a huge amount of interest in recent years because of their crucial functions in maintaining homeostasis through intercellular communication in the lungs.
      miRNAs are endogenous, single-strand, noncoding RNAs, 20 to 23 nucleotides in length, that regulate translation through their interactions with mRNA transcripts.
      • Baek D.
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      • Shin C.
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      The impact of microRNAs on protein output.
      After transcription, miRNAs inhibit gene expression through multiple mechanisms, all of which involve base-specific interactions with target mRNA transcripts. In the human genome, the transcripts of an estimated ~60% of all mRNAs are targeted by miRNAs. miRNAs are first transcribed for the most part by RNA polymerase II as a large primary miRNA, then processed by the endonuclease Drosha into a hairpin structure (precursor miRNA), and then further cleaved by the endonuclease Dicer into a single-strand, mature miRNA.
      • Cullen B.R.
      Transcription and processing of human microRNA precursors.
      • Lee Y.
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      • Han J.
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      The nuclear RNase III Drosha initiates microRNA processing.
      The mature miRNA is incorporated into a complex known as the RNA-induced silencing complex, which contains the proteins Argonaute 2 (Ago-2) and glycine-tryptophan 182 kDa (GW182). As a part of this complex, the mature miRNA regulates gene expression by binding to partially complementary sequences in the 3′-untranslated regions of the target mRNAs, leading to mRNA degradation or translation inhibition.
      • Iorio M.V.
      • Croce C.M.
      MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review.
      A single miRNA may target dozens of mRNAs, and 1 mRNA can be regulated by multiple miRNAs. Currently, there is some evidence that miRNAs are involved in the asthmatic disease process. Some miRNAs reportedly regulate interleukin (IL)-13 and the T helper-2 (Th2) response, which are major components of the asthmatic response.
      • Greene C.M.
      • Gaughan K.P.
      microRNAs in asthma: potential therapeutic targets.
      They are key “fine-tuners” in the pathophysiology of asthma, such as inflammation, smooth muscle hypercontraction, and airway hyper-responsiveness.
      For this review, we chose miRNAs that have been reported to have various roles in cell-to-cell communication and that also had biomarker potential. We summarize the significance of EVs and their miRNAs as new tools for intercellular communication in asthma. We believe that the discovery of EV miRNAs may bring fundamental changes to the understanding and therapeutic strategies of asthma. As mentioned earlier, the term microvesicles has also been used for exosome-like vesicles; clear distinction of exosome and microvesicles has not been adequately established. Therefore, only exosomal miRNAs are considered as EV miRNAs in this review.

      Asthma

      Asthma is a common chronic inflammatory disease involving the respiratory system in which the airways occasionally constrict and become inflamed, often in response to various stimuli, such as allergens, infections, and air pollutants.
      • Martinez F.D.
      • Vercelli D.
      Asthma.
      Asthma is a complex disease made up of disease variants with different underlying pathophysiologic aspects.
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      Asthma endotypes: a new approach to classification of disease entities within the asthma syndrome.
      The World Health Organization estimates that 300 million people are affected by the disease, and that by 2025, another 100 million will have been affected.

      World Health Organization. Chronic respiratory disease—Asthma. http://www.who.int/respiratory/asthma/en/. Accessed [date 28 May 2014].

      Asthma is generally characterized by an abnormal response of the Th2-type CD4+ T lymphocytes against inhaled allergens.
      • Locksley R.M.
      Asthma and allergic inflammation.
      Th2-type CD4+ T lymphocytes modulate allergic disease by secreting a host of proinflammatory cytokines (IL-4, IL-5, and IL-13). The physiologic effects of these ILs result in the majority of the immunologic features of asthma. Airway immune dysfunction in response to inhaled allergens is crucial for the initiation of inflammatory and obstructive responses. Pulmonary inflammation is a major component of asthma involving the influx of mast cells, Th2 cells, and eosinophils. Structural changes leading to airway remodeling, combined with chronic inflammation, give rise to its features, namely, reversible airway hyper-responsiveness and airway obstruction.
      • Seguin R.M.
      • Ferrari N.
      Emerging oligonucleotide therapies for asthma and chronic obstructive pulmonary disease.
      Asthma treatments consist of long- and short-acting β-agonists, leukotriene antagonists, allergen immunotherapy, and inhaled corticosteroids.
      • Ballow M.
      • Akdis C.A.
      • Casale T.B.
      • et al.
      Immune response modifiers in the treatment of asthma: A PRACTALL document of the American Academy of Allergy, Asthma & Immunology and the European Academy of Allergy and Clinical Immunology.
      Inhaled corticosteroids are effective in mild asthma because they improve symptoms and decrease the risk for exacerbation. However, inhaled corticosteroids have important therapeutic limitations in moderate and severe asthma. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and has been associated with adverse effects. In addition, some individuals are corticosteroid resistant.
      There is no way to prevent the initial onset of asthma and no cure for this disorder. The pathophysiologic aspects leading to asthma remain unclear, especially in terms of nonallergic responses, and there are not enough effective therapies. Accordingly, there is a clear need for novel therapeutics, especially for therapy-resistant asthma.

      Functions of miRNAs in Asthma

      miRNAs play important functions in various lung diseases such as lung cancer, interstitial lung diseases, chronic obstructive pulmonary disease, and asthma.
      • Fujita Y.
      • Takeshita F.
      • Kuwano K.
      • Ochiya T.
      RNAi Therapeutic Platforms for Lung Diseases.
      Evidence indicates that miRNAs are a promising technology for current and future therapeutic development in asthma.
      The dysregulated expression of some miRNAs has been found in the airways or lymphocytes of asthmatic patients and an asthmatic murine model. Rodriguez et al
      • Rodriguez A.
      • Vigorito E.
      • Clare S.
      • et al.
      Requirement of bic/microRNA-155 for normal immune function.
      reported that miR-155 is related to the development of inflammatory infiltration into the lung and to airway remodeling. miR-155 targets the transcription factor c-Maf, which promotes IL-4, IL-5, and IL-10 production by Th2 cells. In an experimental study, miR-155–deficient mice developed an altered inflammatory response, with diminished eosinophilic inflammation, reduced eotaxin-2/chemokine (C-C motif) ligand 24 and periostin levels, and reduced Th2 cells.
      • Malmhall C.
      • Alawieh S.
      • Lu Y.
      • et al.
      MicroRNA-155 is essential for T2-mediated allergen-induced eosinophilic inflammation in the lung.
      Other miRNAs have been reported to regulate asthma-like lung inflammation in different murine models. In one model, a toll-like receptor 4–induced Th2 lymphocyte induced high miR-126 expression, and a selective blockade of miR-126 suppressed the asthmatic phenotype.
      • Mattes J.
      • Collison A.
      • Plank M.
      • et al.
      Antagonism of microRNA-126 suppresses the effector function of TH2 cells and the development of allergic airways disease.
      Anti–miR-126 decreased the levels of the Th2 cytokines IL5 and IL-13. Next, high miR-221 expression was detected in the lungs of mice in an ovalbumin-induced asthmatic murine model, and the inhibition of miR-221 reduced inflammation in the airways.
      • Qin H.B.
      • Xu B.
      • Mei J.J.
      • et al.
      Inhibition of miRNA-221 suppresses the airway inflammation in asthma.
      Mayoral et al
      • Mayoral R.J.
      • Deho L.
      • Rusca N.
      • et al.
      MiR-221 influences effector functions and actin cytoskeleton in mast cells.
      suggested that miR-221 affects mast cell–specific phenotypes and contributes to mast cell degranulation and cytokine production in vivo. Finally, in another asthmatic murine model, the downregulation of miR-133a was associated with an increased expression of a small guanosine triphosphatase, RhoA, which led to the augmented contraction and hyper-responsiveness of bronchial smooth muscle cells.
      • Chiba Y.
      • Misawa M.
      MicroRNAs and their therapeutic potential for human diseases: MiR-133a and bronchial smooth muscle hyperresponsiveness in asthma.
      Those studies have presented a functional connection between miRNA expression and asthma pathogenesis, and they suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Although further investigations regarding the role of miRNAs in relation to the pathogenesis of asthma are required, viewing miRNAs as key players in gene expression may contribute to the development of a novel treatment for asthma.

      Function of Extracellular Vesicles in Asthma

      The first evidence of EV function was reported by immunology researchers. In 1996, Raposo et al
      • Raposo G.
      • Nijman H.W.
      • Stoorvogel W.
      • et al.
      B lymphocytes secrete antigen-presenting vesicles.
      found that B lymphocyte–derived EVs function as immune system activators. After some groups demonstrated that dendritic cell (DC)-derived EVs modulated immune reactions by activating T and B lymphocytes,
      • Zitvogel L.
      • Regnault A.
      • Lozier A.
      • et al.
      Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes.
      • Skokos D.
      • Le Panse S.
      • Villa I.
      • et al.
      Mast cell-dependent B and T lymphocyte activation is mediated by the secretion of immunologically active exosomes.
      important studies in the field of asthma immunology were initiated.
      EVs are released from several cells that may be involved in allergies, including mast cells, DCs, T cells, and bronchial epithelial cells in the lungs. For example, mast cell–derived EVs highly induce DC maturation. Furthermore, DC-derived EVs can transport allergens and activate allergen-specific Th2 cells.
      • Admyre C.
      • Telemo E.
      • Almqvist N.
      • et al.
      Exosomes - nanovesicles with possible roles in allergic inflammation.
      It is important to understand the function of various EV types in the lungs. In humans, EVs have been found in bodily fluids, including serum, urine, and BALF.
      • Admyre C.
      • Telemo E.
      • Almqvist N.
      • et al.
      Exosomes - nanovesicles with possible roles in allergic inflammation.
      EVs can exert their effects by delivering their components, such as proteins, mRNAs, and miRNAs, to recipient cells. However, the function of EVs in modulating cell-to-cell communication in the context of lung diseases is thus far unexplored. To date, some studies have suggested that EVs in BALF have important roles in allergic reactions and in the immunologic regulation of asthma
      • Torregrosa Paredes P.
      • Esser J.
      • Admyre C.
      • et al.
      Bronchoalveolar lavage fluid exosomes contribute to cytokine and leukotriene production in allergic asthma.
      • Admyre C.
      • Grunewald J.
      • Thyberg J.
      • et al.
      Exosomes with major histocompatibility complex class II and co-stimulatory molecules are present in human BAL fluid.
      . These data also demonstrated that EVs have several functions in the progression of allergic reactions. In contrast, studies also have reported that tolerizing EVs could block an allergic response or prevent allergy development.
      In 2003, Admyre et al
      • Admyre C.
      • Grunewald J.
      • Thyberg J.
      • et al.
      Exosomes with major histocompatibility complex class II and co-stimulatory molecules are present in human BAL fluid.
      reported for the first time that EVs are present in the BALF of humans and that they might have a regulatory function in local immunologic defense. The EVs from DCs express major histocompatibility complex classes I and II in addition to co-stimulatory molecules on their surfaces, and they can induce the antigen-specific activation of T cells. Prado et al
      • Prado N.
      • Marazuela E.G.
      • Segura E.
      • et al.
      Exosomes from bronchoalveolar fluid of tolerized mice prevent allergic reaction.
      reported that EVs from the BALF of tolerized mice could be given intranasally to prevent allergic sensitization. These tolerogenic EVs inhibited the classic pathology related to allergies, such as the immunoglobulin E response, Th2 cytokine production, and airway inflammation. The investigators proposed that EV-based vaccines could represent an alternative to conventional therapy for allergic diseases. Furthermore, it has been reported that EVs from the BALF of patients with asthma could contribute to subclinical inflammation by increasing IL-8 and leukotriene C4 generation in bronchial epithelial cells, and that the use of cysteinyl leukotriene receptor 1 antagonist montelukast was associated with reduced EV-induced IL-8 secretion.
      • Torregrosa Paredes P.
      • Esser J.
      • Admyre C.
      • et al.
      Bronchoalveolar lavage fluid exosomes contribute to cytokine and leukotriene production in allergic asthma.
      Those investigators reported that EVs from the BALF of asthmatic patients could increase IL-8 and leukotriene production in bronchial epithelial cells in vitro. They also speculated that EVs from the analyzed BALF partly originate from bronchial epithelial cells because of their expression of the epithelial marker mucin 1 in combination with human leukocyte antigen DR. However, the cell types in the lungs that were related to EV-mediated cross-talk and their paracrine effects remain unclear.
      In 2013, Kulshreshtha et al
      • Kulshreshtha A.
      • Ahmad T.
      • Agrawal A.
      • Ghosh B.
      Proinflammatory role of epithelial cell-derived exosomes in allergic airway inflammation.
      demonstrated that bronchial epithelial cells are the main providers of EVs in the lungs of asthmatic patients. Those investigators reported that EV marker proteins were mostly localized to bronchial epithelial cells and macrophages in lung tissue sections. As a novel finding of EV-mediated paracrine effects, IL-13–treated bronchial epithelial cell–derived EVs promote the proliferation of undifferentiated macrophages. Furthermore, the reduction in EV secretion by GW4869 (an inhibitor of EV production) are reported to ameliorate asthmatic features in an asthmatic murine model. Those data indicate the potential contributions of epithelial cell-derived EVs to the pathogenesis of asthma. These findings could have implications in the development of future treatments targeting the inhibition of EV secretion by cutting off EV-mediated cross-talk in patients with asthma and other inflammatory lung diseases.

      Intercellular Communication with EV miRNAs in Asthma

      To date, studies about EV miRNAs have provided considerable insight into the undefined mechanisms underlying various biological phenomena. A recent development in the EV field was provided by an article published in 2007. For the first time, Valadi et al
      • Valadi H.
      • Ekstrom K.
      • Bossios A.
      • et al.
      Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.
      reported that EVs derived from human and murine mast cell lines transported RNA to another mast cell, which was then translated, indicating that the transferred RNA was biologically active. In 2010, 3 groups independently reported that EV miRNAs are secreted and transported between cells and that these miRNAs promoted RNA interference effects in the recipient cells.
      • Kosaka N.
      • Iguchi H.
      • Yoshioka Y.
      • et al.
      Secretory mechanisms and intercellular transfer of microRNAs in living cells.
      • Pegtel D.M.
      • Cosmopoulos K.
      • Thorley-Lawson D.A.
      • et al.
      Functional delivery of viral miRNAs via exosomes.
      • Zhang Y.
      • Liu D.
      • Chen X.
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      Secreted monocytic miR-150 enhances targeted endothelial cell migration.
      Pegtel et al
      • Pegtel D.M.
      • Cosmopoulos K.
      • Thorley-Lawson D.A.
      • et al.
      Functional delivery of viral miRNAs via exosomes.
      reported that miRNAs of viral origin were found in EVs secreted by infected cells and that their transfer to noninfected cells led to the regulation of some of target genes of these miRNA. Our group demonstrated that a tumor-suppressive miRNA travels between 2 types of cells and promotes cell growth inhibition.
      • Kosaka N.
      • Iguchi H.
      • Yoshioka Y.
      • et al.
      Secretory mechanisms and intercellular transfer of microRNAs in living cells.
      In immunology research, EV miRNAs have also reportedly played important functions.
      • Chaput N.
      • Thery C.
      Exosomes: immune properties and potential clinical implementations.
      • Xu L.
      • Yang B.F.
      • Ai J.
      MicroRNA transport: a new way in cell communication.
      For example, EV miRNA transfer demonstrates a novel mechanism of DC-to-DC communication.
      • Montecalvo A.
      • Larregina A.T.
      • Shufesky W.J.
      • et al.
      Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes.
      Mature DC-derived EVs have higher expression levels of miRNAs related to proinflammatory transcripts compared with immature DC-derived EVs. Mittelburn et al
      • Mittelbrunn M.
      • Gutierrez-Vazquez C.
      • Villarroya-Beltri C.
      • et al.
      Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells.
      reported the existence of an antigen-driven unidirectional transfer of miRNAs from the T cell to the antigen-presenting cell. These data suggest that miRNAs transferred during immune synapsis can regulate gene expression in recipient cells.
      These findings support the idea that EVs may serve as an intercellular transporter of miRNAs for cell-to-cell communication in the lungs. Although it is well-established that EV-mediated cellular communication can influence disease phenotypes, the function of individual contents within EVs in lung diseases is still unknown. To date, the EV miRNA contents caused by asthma have not been described well. In 2013, Levänen et al
      • Levanen B.
      • Bhakta N.R.
      • Torregrosa Paredes P.
      • et al.
      Altered microRNA profiles in bronchoalveolar lavage fluid exosomes in asthmatic patients.
      reported for the first time that miRNAs from EVs in the BALF of asthmatic patients are different from those in healthy controls. EV RNA was analyzed by using microarrays, and selected findings were validated by real-time polymerase chain reaction. Subsequently, the levels of 24 types of miRNA were significantly different between asthmatic patients and controls. The altered miRNAs were primarily downregulated, and the report suggests that a number of them may be involved in the regulation of IL-13 by pathway analysis. Remarkably, the expression profile of the altered miRNAs was significantly correlated with pulmonary function in the asthmatic group. Thus, EV miRNAs may differentiate between asthma and controls.
      In the near future, studies of these miRNAs will provide novel insights into cell-to-cell communication by EVs in the lungs. Additionally, it is necessary to carefully analyze the EV miRNA-mediated cross-talk between different cell types in the lungs. We believe that EV miRNAs play important roles in lung biology and are key players in the cell-to-cell communication in the lungs (Figure 2).
      Figure thumbnail gr2
      Figure 2The function of extracellular vesicles (EVs) in asthma. EVs have been isolated and characterized from bronchoalveolar lavage fluid. EVs are released from several cells that have been implicated in allergies, including mast cells, dendritic cells, T cells, and bronchial epithelial cells in the lungs. The potential contributions of bronchial epithelial cell–derived EVs to asthma have been reported. EV-mediated cross-talk between different cells in the lungs may regulate asthma pathogenesis.

      Circulating miRNAs as Potential Biomarkers for Asthma

      Intercellular miRNAs provide important functions in many biological processes. As described earlier, recent data have shown that miRNAs are present in the extracellular spaces, such as BALF, blood, urine, and saliva.
      • Kosaka N.
      • Iguchi H.
      • Ochiya T.
      Circulating microRNA in body fluid: a new potential biomarker for cancer diagnosis and prognosis.
      This finding suggests that the miRNAs are potential noninvasive biomarkers for numerous diseases and conditions.
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      • Parkin R.K.
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      • et al.
      Circulating microRNAs as stable blood-based markers for cancer detection.
      • Hanke M.
      • Hoefig K.
      • Merz H.
      • et al.
      A robust methodology to study urine microRNA as tumor marker: microRNA-126 and microRNA-182 are related to urinary bladder cancer.
      • Park N.J.
      • Zhou H.
      • Elashoff D.
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      Salivary microRNA: discovery, characterization, and clinical utility for oral cancer detection.
      • Cortez M.A.
      • Bueso-Ramos C.
      • Ferdin J.
      • et al.
      MicroRNAs in body fluids--the mix of hormones and biomarkers.
      BALF is a useful research tool, but it is not suitable for clinical monitoring. In this context, serum or urine miRNAs are promising biomarkers in clinical settings. Some data support that serum miRNAs are suitable biomarkers for various cancers, inflammation, and allergies.
      • Weiland M.
      • Gao X.H.
      • Zhou L.
      • Mi Q.S.
      Small RNAs have a large impact: circulating microRNAs as biomarkers for human diseases.
      The studies cited previously support the existence of miRNA-containing EVs; however, they may not be the most prevalent form of circulating miRNAs. miRNAs may be secreted by EVs and by protein-miRNA complexes, such as high-density lipoprotein (HDL) and the Ago-2 protein, which is a part of the RNA-induced silencing complex.
      • Redis R.S.
      • Calin S.
      • Yang Y.
      • et al.
      Cell-to-cell miRNA transfer: from body homeostasis to therapy.
      Arroyo et al
      • Arroyo J.D.
      • Chevillet J.R.
      • Kroh E.M.
      • et al.
      Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma.
      reported that the majority of serum miRNAs are present as Ago-2–miRNA complexes, but not within EVs. Significantly, only EV miRNAs reportedly have a function in communicating between cells and play a role in immune responses, including asthma.
      It was recently reported that EV miRNAs in exhaled breath have potential as biomarkers of pulmonary diseases.
      • Sinha A.
      • Yadav A.K.
      • Chakraborty S.
      • et al.
      Exosome-enclosed microRNAs in exhaled breath hold potential for biomarker discovery in patients with pulmonary diseases.
      • Pinkerton M.
      • Chinchilli V.
      • Banta E.
      • et al.
      Differential expression of microRNAs in exhaled breath condensates of patients with asthma, patients with chronic obstructive pulmonary disease, and healthy adults.
      miRNAs have been detected in exhaled breath condensates, where 11 miRNAs were found to be expressed differently in asthmatic patients compared with healthy controls. Exhaled breath condensate is emerging as an important source of biomarkers that can be obtained noninvasively.
      • Sinha A.
      • Yadav A.K.
      • Chakraborty S.
      • et al.
      Exosome-enclosed microRNAs in exhaled breath hold potential for biomarker discovery in patients with pulmonary diseases.
      • Pinkerton M.
      • Chinchilli V.
      • Banta E.
      • et al.
      Differential expression of microRNAs in exhaled breath condensates of patients with asthma, patients with chronic obstructive pulmonary disease, and healthy adults.
      These data provide a proof of principle that airway miRNAs in exhaled breath represent a fertile field for clinical biomarkers.

      Conclusions

      With the amazing growth in the number of EV studies in recent years, it is clear that the intercellular messenger function of EVs now constitutes an exciting field. Investigating the biological functions of EVs is an emerging and rapidly progressing area in lung disease biology. Data from the studies cited in this review suggest that miRNAs play a significant role in the pathogenesis of asthma by the degradation of target mRNAs or by the inhibition of translation. Therefore, miRNA-based medicines for asthma could have the potential to treat the inflammation and hyper-responsiveness of this disease. Over the next few years, EV miRNA studies will provide remarkable insights into asthma. This field of research holds great potential for therapeutic applications relating to asthma. In addition, circulating miRNAs are potential noninvasive biomarkers. Future studies will reveal whether serum and airway miRNAs can be used as specific biomarkers to distinguish disease severity and endotypes. We emphasize that it is important to investigate the role of EV miRNAs in cell-to-cell communication and to explore the effectiveness of these molecules as biomarkers in humans.

      Conflicts of Interest

      This work was supported in part by a grant-in-aid for the Third-Term Comprehensive 10-Year Strategy for Cancer Control of Japan; Project for Development of Innovative Research on Cancer Therapeutics (P-Direct); Scientific Research on Priority Areas Cancer, Scientific Research on Innovative Areas (functional machinery for non-coding RNAs) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology; the National Cancer Center Research and Development Fund (23-A-2, 23-A-7, and 23-C-6); the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation; the Project for Development of Innovative Research on Cancer Therapeutics; and the Japan Society for the Promotion of Science through the Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program) initiated by the Council for Science and Technology Policy.
      The authors have indicated that they have no conflicts of interest with regard to the content of this article.

      Acknowlodgments

      Dr. Fujita and Dr. Ochiya responsible to the concept of the review. Dr. Fujita responsible for the writing of the first draft of the manuscript. Dr. Yoshioka was responsible for assisting with sudy selection. Dr. Yoshioka, Drs. Ito, Drs. Araya, Drs. Kuwano and Dr. Ochiya were responsible for the review and critical comment of the manuscript.

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