Extracellular Vesicle-Shuttling MicroRNAs Regulate the Development of Inflammatory Lung Responses

Abstract

MicroRNAs are small single-stranded, non-coding RNAs which have a known role in post-transcriptional regulation of gene expression. Recent studies have reported that extracellular vesicles are capable of specific delivery of miRNAs to a target cell or tissue from a host cell. MiRNAs are generated by host cells, selectively packaged into EVs, and then delivered to nearby target cells with full functionality. After delivery to the target cells, these EV-packaged miRNAs regulate the translation of their target genes. Thus, EV transported miRNAs have become a newly understood method for intercellular communication. In this review, we summarize the novel findings of EV-miRNA transfer in acute lung injury, chronic obstructive pulmonary disease, bronchopulmonary dysplasia, asthma, and idiopathic pulmonary fibrosis.

INTRODUCTION

MiRNA Function

Since the first discovery of non-coding RNAs (ncRNAs) in 1993, researchers have worked to uncover the role ncRNAs play in cells [8]. MicroRNAs (miRNAs), which are a type of ncRNA, are small (about 22 nucleotides) single stranded RNAs, which play a critical role in posttranscriptional regulation of protein expression [1]. MiRNAs are encoded and transcribed inside the nucleus, and are then transported to the cytoplasm where they are incorporated into the ribonucleoprotein-silencing machinery [3]. Through this regulatory role, miRNAs target messenger RNAs (mRNAs) for either suppression or degradation, which leads to an overall decrease in the expression of the gene translated from that mRNA [2]. In recent years, studies have proved that miRNAs are likely involved in most cell processes, and have a major impact in many diseases, including vascular inflammation [4,9].

EV Function

Extracellular vesicles (EVs) are membranous structures which consist of exosomes and macrovesicles originating from the endosomal system, as well as apoptotic bodies originating from apoptotic cells [5]. EVs are established as a method of cell to cell communication, which allows for the passing cellular material between host and target cell [6]. Release of EVs was originally thought to be a method of discarding membranous proteins from host cells, however, recent studies have reported evidence that intercellular communication through EVs plays an important role in the physiological and pathological processes of multiple diseases [7]. The type and amount of EVs released vary based on the status of the disease, and therefore, EVs could serve as novel biomarkers for various lung diseases [40]. With the vital discovery of EVs and research into their role in multiple diseases, EVs have the potential to become a new drug delivery system and a novel diagnostic / therapeutic target [41].

EVs are known to contain DNA, RNA, and proteins, which are passed from host cell to target cell [42]. Despite the various components found in EVs, which may all play a regulatory role on disease biogenesis, we will focus specifically only on EV-miRNAs in this review.

Discovery of EV-miRNA

EV miRNAs are considered to be possible diagnostic markers and therapeutic targets of multiple pulmonary diseases [17]. Previous studies have shown that not all miRNAs are transferred via exosomes, and that there is a selectivity mechanism which determines the regulatory miRNAs chosen for export [16]. The first to report of EV mediated miRNA transfer between cells was in 2007 by Valadi et al. [21]. Following this report, in 2010, three independent studies reported that these EV transferred miRNAs created an RNA interference effect in the recipient cell [22,23,24]. Since these findings, there have been multiple papers suggesting EV-containing miRNAs play crucial regulatory roles in many pulmonary diseases including ALI, COPD, asthma, pulmonary arterial hypertension (PAH), and pulmonary fibrosis [7,31].

Current Studies of EV-miRNAs in Lung Inflammation

Major categories of EVs

The three major categories of EVs are commonly referred to as; exosomes, microvesicles, and apoptotic vesicles [10,14]. Exosomes have an endosomal origination, are 40–150nm in size, and are involved in intercellular communication [11]. Exosomes are formed by the invagination of the cells lipid bilayer, followed by the release from within the cell after acquiring proteins, DNA, and RNA from their cell of origin [11,12]. Exosome release occurs by the fusion of the late exosome with the cell membrane [12]. Microvesicles originate from the plasma membrane, are 50–2000nm in size, and transfer proteins and nucleic acids to nearby cells [25]. Unlike exosomes, microvesicles are formed by the outward budding and splitting of the plasma membrane [25,26]. Apoptotic vesicles are released by the disassembly of apoptotic cells, and are 1,000–5,000 nm in size [10]. In recent years, there has been increasing evidence which suggests miRNA-containing EVs produced during apoptosis play a significant immune regulatory role in multiple diseases [27].

EV miRNAs in Acute Lung Injury

Acute Respiratory Distress Syndrome (ARDS) is a devastating syndrome responsible for significant morbidity and mortality. Its mild form is known as Acute Lung Injury (ALI). Non-cardiogenic pulmonary edema, vascular leakage, inflammation, lung epithelial cell injury and dysfunction play important roles in the pathogenesis of ARDS. To date, very few studies have been conducted to study the transfer of miRNAs by EVs in the pathophysiology of lung inflammation. In ALI, the EV transfer of miR-146a and miR-155 have both been reported in cells derived from dendritic cells [13,15]. In the study of the EV transfer of miR-146a and miR-155 by Alexander et al., it was found that miR-146a inhibits endotoxin induced inflammation, while miR-155 promotes this inflammation [13,15]. In our previous study, we found that Microvesicles (MVs) were the main type of EV found in the early stages of hyperoxia [28]. In this study, we also identified the induction of both miR-320a and miR-221 in epithelial-derived MVs released in response to hyperoxia [28]. The delivery of these specific miRNAs (miR-320a and miR-221) via MVs to target cells promotes macrophage-mediated pro-inflammatory effects [28]. Another report, which studied EVs isolated from Broncho-Alveolar Lavage Fluid (BALF) of patients with influenza virus-derived ARDS, reported the upregulation of EVs containing miRNA-17–5p [42]. The study concluded that the EV delivered miR-NA-17–5p lead to a downregulation of antiviral factor M×1 and enhanced viral replication in influenza virus-derived ARDS patients [42].

EV miRNAs in COPD

Chronic obstructive Pulmonary Disease (COPD) is a respiratory disease characterized by inflammation of the airways leading to the lungs [18]. To date, there is major lack in reports studying EV-miR-NAs in COPD. A study by Fuijita et al. found that the EV transfer of HBEC-derived miR-210 plays a role in the biogenesis of COPD [19]. This study found that specifically, miRNA-210 regulates autophagy processes by targeting ATG7, and that miRNA-210 expression levels were inversely correlated with ATG7 expression in lung fibroblasts (LFs). An additional study found that cigarette smoke induced the EV transfer of miR-191, miR-126, and miR-125a via ceramide-synthesis enzyme acid sphingomyelinase (aSMase) [20]. These miR-NAs were transferred to macrophages, leading to the promotion of apoptotic cells [20].

EV miRNAs in Bronchopulmonary dysplasia

Bronchopulmonary Dysplasia (BPD) is a form of chronic lung disease which is commonly seen in premature babies [32]. Despite the increase in study of the disease, the rate of complications is still remarkably high, and no safe therapy developed has had a major impact on the rate of incidence and severity of BPD [36]. One study, which focused on characterizing the change in EV miRNA expression in BPD patients, tested human samples to observe the different expressions of EV miRNAs in human mesenchymal stem cell-derived extracellular vesicles (mEVs) vs fibroblast EVs (fEVs) [37]. This study discovered 30 miRNAs that were expressed notably greater in mEVs than fEVs [37]. Of these thirty EV miRNAs, the study concluded miR-1246, miR-6511a-5p, and miR-22–3p as the top 3 differentially expressed in mEVs, and the top 3 miRs from mesenchymal stem cells (MSCs) sorted into mEVs were miR-630, miR-4286, and miR-4454+7975 [37]. MSC-derived mEVs have been reported to reduce tissue fibrotic responses [38]. Further investigation into the regulatory mechanisms of EV miRNAs in BPD could offer a reliable therapy to treating this poorly understood disease.

EV miRNAs in Asthma

Asthma is a chronic inflammatory disease of the airways, and patients who have asthma usually show symptoms of wheezing, dyspnea, chest tightness, and cough [34]. Further studies into asthma will be required to broaden our knowledge of the disease and understand the complex mechanisms involved in the biogenesis [33]. The first investigation of EV miRNAs in asthma studied the expression levels of a wide range of miRNAs from BALF-EVs isolated in asthmatic patients [35]. This study by Levanen et al. identified 24 miRNAs (let-7c, let-7b, 141, 200b, let-7d, let-7a, 21, 27, let-7e, 34c-5p, 34b-5p, 19b, 1972, 665, 658, 483–5p, 0022, 0024, 0026a, 0099a, 0200c, 1268, 0203, 0130a) were significantly altered between control and asthma patients [35]. This investigation did not look further into the mechanisms of these EV miRNAs in asthmatic patients, but concluded that these findings suggest some, if not all of these reported EV miRNAs play a role in the regulation of asthma pathology in the lung [35].

EV miRNAs in Idiopathic Pulmonary Fibrosis

Idiopathic Pulmonary Fibrosis (IPF) is a pulmonary disease which leads to progressive decline of lung function, characterized by chronic inflammation [29]. Despite previous success in the molecular diagnostics and pathobiology of IBF, the biogenesis of IPF is still unclear, and requires further study [30]. One study published in 2016, identified the promotion of serum EV-containing miRNA-21–5p expression in IPF mice and patients [31]. The study did not map the direct regulatory effect of EV miRNA-21–5p, however they did suggest a novel biomarker, EV miRNA-21–5P, which could be used clinically to distinguish patients who require intensive IBF therapy [31]. Another study, which studied mEV miRNAs in human bone marrow-derived MSCs, which target profibrotic genes in IPF fibroblasts discovered the upregulation of mEV packaged miR-199a-3p, 21–5p, 630, 22–3p, 196–5p, 199b-5p, 34a-5p and 148a-3p [37]. Previous studies have identified the roles of these miRNAs in IPF fibroblasts: serum miR-21 in EV is related to poor prognosis in IPF, and miR-21 activates myofibroblasts in vitro; miR-199 is associated in liver fibrosis, and is also upregulated in IPF and activates myofibroblasts; miR-22 suppresses cardiac fibrogenesis and cirrhosis; miR-196–5p suppresses renal fibrosis; and miR-34–5p is pro-fibrogenic in the heart and controls pneumocyte senescence in IPF [37]. This study also reports that miR-630, which the study suggests may regulate adherens-junction dependent cell migration and fibroblast attack, is the most upregulated mEV packaged miRNA in their study [37]. These Thy-1-mediated Mev-containing miRNAs seem to work in unison to induce an anti-myofibroblastic effect in IPF fibroblasts [37].

Pitfalls of Current EV-miRNA Research and Future Directions

In addition to the lack of reports studying EV-miRNAs in lung inflammation, the papers which have been published, do possess clear pitfalls. The majority of published reports focus on the potential of EVs or EV-miRNAs as biomarkers, however, the physiological and pathological functions these EV-miRNAs are still unexplored. Additionally, of those reports which do study the physiological functions of these EV-miRNAs, there are still critical issues which need further clarification. The concentration of each specific miRNA in the EVs must be determined to understand the critical threshold necessary to trigger a downstream function in the recipient cell. Moreover, given that each EV may only carry a limited number of miRNAs, it should be determined how many EVs are required to trigger the functional effects in the recipient cells. It also is critical to determine if there is a mechanism which guides EVs to specific recipient cells. For example, in our studies, all the macrophage-derived or epithelial cell-derived EVs target macrophages, followed by the same cells with their “parent” cells [28]. Interestingly, only macrophages engulf the EVs, but not so much by polymorphonuclear cell family (PMNs). Both macrophages and PMNs are phagocytes, therefore, phagocytosis cannot explain the uptake entirely and some other mechanisms must exist. Lastly, there is no consistent manner to block EV generation. All the functional studies in the above published work have some degree of “artifacts” which likely deliver an extreme (over-dosed) amount of EVs and EV-miR-NAs when doing in vivo functional studies. All of the above pitfalls should be corrected and studied in future studies of EV-miRNAs.

Future studies into EV-miRNAs will help to uncover the unclear role of EVs and EV-miRNAs in the pathogenesis of many diseases. Firstly, future studies should look to reveal the detailed mechanisms of how miRNAs are selectively encapsulated into EVs. Secondly, work is necessary to further understand the detailed signaling pathways of EV-miRNAs after being taken by the recipient cells. Additionally, it is critical for the development of a consistent and fast method to detect EV-miRNAs in each disease models, using body fluids. Lastly, a classification system should be created for microvesicles, exosomes and apoptotic bodies; specifically, by their function, generation, markers and physiological significance.

Figure 1:

Schema of the three major categories of Extracellular vesicles (EVs)

Figure 2:

Schema of the function of EV-miRNAs.

Table 1:

Currently reported EV-miRNAs involved in lung inflammation.

Disease	EV-miRNA	Origin	Type of EV	Author
ALI	miR-146a, 155	Dendritic cells	Exosome	Zeng 2013, Alexander 2015
ALI	miR-320a, 221	Epithelial cells	Microvesicles	Lee 2016
ALI	miRNA-17-5p	Broncho-alveolar lavage fluid	Total EVs	Scheller 2018
COPD	miR-210	Human bronchial epithelial cells (HBEC)	Total EVs	Fujita 2015
COPD	miR-191, 216, 215a	Lung endothelial cells	Exosome	Serban 2016
BPD	miR-1246, 6511a5p, 22-3p, 21-5p, 296-5p, 196a-5p, 708-5p, 4707, 1244-5p, 20a/b5p, 214-3p, 148a3p, 199b-5p, 196-3p+6732-5p, 199a/b-3p, 6715p, 34a-5p, 648, 515-5p, 181a-23p, 630, 518e-3p, 6503-3p, 887-3p, 10b-5p, 1269a, 1915-3p, 193a/ b5p, 644a, 518d-3p	Mesenchymal	Total EVs	Shentu 2017
Asthma	miR-let-7c, let-7b, 141, 200b, let-7d, let-7a, 21, 27, let-7e, 34c-5p, 34b-5p, 19b, 1972, 665, 658, 483-5p, 0022, 0024, 0026a, 0099a, 0200c, 1268, 0203, 0130a	BALF	Exosome	Levanen 2013
IPF	miR-21-5p	Serum	Total EVs	Makiguchi 2016
IPF	miR-199a-3p, 215p, 630, 22-3p, 196-5p, 199b-5p, 34a-5p, 148a-3p	Bone marrowde-rived MSCs	Total EVs	Shentu 2017