Bronchoalveolar Lavage Fluid Exosomes Research Articles

CircRNAs in BALF exosomes and plasma as diagnostic biomarkers in patients with acute respiratory distress syndrome caused by severe pneumonia.

The transcriptomic studies targeting circular RNAs (circRNAs) in bronchoalveolar lavage fluid (BALF) exosomes of acute respiratory distress syndrome (ARDS) patients caused by severe pneumonia have rarely been reported. This study aimed to screen and validate abnormally expressed circRNAs in exosomes from BALF of patients with ARDS caused by severe pneumonia and then evaluate the diagnostic values of these circRNAs for ARDS. BALF was collected from four patients with ARDS caused by severe pneumonia and four healthy subjects. CircRNA expression profile was obtained by microarray analysis in BALF exosomes of the discovery cohort. The differentially expressed circRNAs in BALF exosomes were verified by real-time quantitative PCR (RT-qPCR) and underwent competitive endogenous RNA (ceRNA) network construction and functional enrichment analysis. A total of 629 circRNAs were differentially expressed in BALF exosomes between ARDS patients and healthy subjects. Nine differentially expressed circRNAs were validated by RT-qPCR, and seven were consistent with the results of microarray analysis. CeRNA network analysis was performed for hsa_circRNA_002809, hsa_circRNA_042882, and hsa_circRNA_104034. Functional enrichment analysis showed that the target genes were mainly associated with hypoxia-induced damage, inflammatory response, and the HIF-1 signaling pathway. Hsa_circRNA_042882 and hsa_circRNA_104034 can be regarded as promising diagnostic biomarkers for patients with ARDS caused by severe pneumonia, with remarkable sensitivity and specificity of the area under the curve of 0.8050 and 1 or 0.835 and 0.799, respectively. This study obtained circRNA expression profiles of ARDS patients, and hsa_circRNA_042882 and hsa_circRNA_104034 were regarded as promising diagnostic biomarkers for patients with ARDS caused by severe pneumonia.

MiR-223-3p-loaded exosomes from bronchoalveolar lavage fluid promote alveolar macrophage autophagy and reduce acute lung injury by inhibiting the expression of STK39.

This study investigated the molecular mechanism by which bronchoalveolar lavage fluid exosomes (BALF-exo) alleviated acute lung injury (ALI). BALF-exo was isolated from mice. LPS was used to induce inflammation in rat alveolar macrophages (NR8383). NR8383 cell models were treated with BALF-exo or BALF-exo loaded with miR-223-3p mimics/inhibitors, or STK39 was overexpressed in NR8383 cells before LPS and BALF-exo treatment. These cells were subjected to apoptosis, autophagy, and inflammation assays. RNA immunoprecipitation and dual-luciferase reporter assay were conducted to verify the binding between miR-223-3p and STK39. LPS-induced ALI mouse models were treated with BALF-exo loaded with miR-223-3p mimics. miR-223-3p was lowly expressed in BALF-exo from LPS-treated mice. BALF-exo loaded with miR-223-3p mimics increased viability and autophagyand decreased apoptosis and inflammation in NR8383 cell models. Inhibition of miR-223-3p in BALF-exo or overexpression of STK39 in NR8383 cells repressed the therapeutic effect of BALF-exo in LPS-treated NR8383 cells. STK39 was a target gene of miR-223-3p. miR-223-3p shuttled by BALF-exo negatively regulated the expression of STK39 in NR8383 cells. BALF-exo loaded with miR-223-3p mimics also reduced lung injuries in ALI mice. In conclusion, miR-223-3p loaded in BALF-exo alleviates ALI by targeting STK39 in alveolar macrophages.

Clinical study on the relationship between the exosomes in bronchoalveolar lavage fluid and plasma and the severity of lung injury and outcome in early acute respiratory distress syndrome patients

Objective: To investigate the relationship between the levels of exosomes in bronchoalveolar lavage fluid (BALF) and plasma and the severity of lung injury and its outcome in patients with acute respiratory distress syndrome (ARDS). Methods: Patients who were admitted to the Department of Critical Care Medicine, Zhongda Hospital Affiliated to Southeast University and received invasive mechanical ventilation were selected from August 2020 to April 2021, and they were divided into ARDS group and non-ARDS group. Finally, 33 ARDS patients were included, including 18 males and 15 females, aged (65.5±15.5) years; 10 non-ARDS patients, 8 males and 2 females, aged (57.2±15.3) years. The BALF and plasma of the two groups of patients were collected within 24 hours after enrollment, and the total exosomes of the samples were collected by ultracentrifugation. Nanoparticle tracking analysis (NTA) was used to detect and compare the differences in exosome content between the two groups. Correlation of content with the severity and prognosis of lung injury in ARDS patients. Results: There was no significant difference in gender and age between ARDS group and non-ARDS group (both P>0.05). The exosome in plasma of ARDS group was significantly higher than that of non-ARDS group [(25.3±1.2)/ml vs (24.2±1.6)/ml, P=0.031], while the exosomes in BALF of ARDS group was also higher than that of non-ARDS group [(26.5±1.6)/ml vs (24.6±1.1)/ml, P=0.001]. The exosomes in BALF of patients with ARDS caused by intrapulmonary causes was higher than that in ARDS group caused by extrapulmonary causes [(26.9±1.5)/ml vs (25.2±0.9)/ml, P=0.01], and the infection caused by bacterial shows that the highest exosome level in BALF. The exosomes in the BALF of the mild ARDS group was significantly lower than that of the severe ARDS group [(25.7±1.3)/ml vs (27.2±1.5)/ml, P=0.038]; the exosomes in BALF of ARDS patients was negatively correlated with P/F ratio (r=-0.38, P=0.03); and it was positively correlated with Murray lung injury score (r=0.47, P=0.01). However, the static compliance levels, length of hospital stay, duration of mechanical ventilation, and 28-day outcome were not associated with the exosomes in BALF. Conclusion: Compared with non-ARDS patients, ARDS patients have significantly higher levels of exosomes in BALF and plasma, there is a certain correlation between exosomes derived from BALF and the severity of lung injury in ARDS.

The role of exosomes from BALF in lung disease.

Exosomes are released from a variety of immune cells and nonimmune cells, the phospholipid vesicle bilayer membrane structure actively secreted into tissues. Recently, exosomes were demonstrated to be effectively delivered proteins, cholesterol, lipids, and amounts of DNA, mRNA, and noncoding RNAs to a target cell or tissue from a host cell. These can be detected in blood, urine, exhaled breath condensates, bronchoalveolar lavage fluid (BALF), ascites, and cerebrospinal fluid. BALF is a clinical examination method for obtaining alveolar cells and biochemical components, reflecting changes in the lungs, so it is also called liquid biopsy. Exosomes from BALF become a new method for intercellular communication and well‐documented in various pulmonary diseases. In chronic obstructive pulmonary disease (COPD), BALF exosomes can predict the degree of COPD damage and serve as an effective monitoring indicator for airflow limitation and airway remodeling. It also mediates antigen presentation in the airways to the adaptive immune system as well as costimulatory effects. Furthermore, BALF exosomes from acute lung injury and infective diseases are closely related to various infections and lack of oxygen status. BALF exosomes play an important role in the diagnosis and prognosis of lung cancer. The effect of immunomodulatory role for BALF exosomes in adaptive and innate immune responses has been studied in sarcoidosis. The intercellular communication in the microenvironment of BALF exosomes in pulmonary fibrosis and lung remodeling have been studied. In this review, we summarize the novel findings of exosomes in BALF, executed function by protein, miRNA, DNA cytokine, and so on in several pulmonary diseases.

Sarcoidosis exosomes stimulate monocytes to produce pro-inflammatory cytokines and CCL2

Pulmonary sarcoidosis has unknown etiology, a difficult diagnostic procedure and no curative treatment. Extracellular vesicles including exosomes are nano-sized entities released from all cell types. Previous studies of exosomes from bronchoalveolar lavage fluid (BALF) of sarcoidosis patients have revealed pro-inflammatory components and abilities, but cell sources and mechanisms have not been identified. In the current study, we found that BALF exosomes from sarcoidosis patients, but not from healthy individuals, induced a dose-dependent elevation of intracellular IL-1β in monocytes. Analyses of supernatants showed that patient exosomes also induced release of IL-1β, IL-6 and TNF from both PBMCs and enriched monocytes, suggesting that the observed effect is direct on monocytes. The potently chemotactic chemokine CCL2 was induced by exosomes from a subgroup of patients, and in a blocking assay the exosome-induced CCL2 was reduced for 13 out of 19 patients by the asthma drug Montelukast, a cysteinyl leukotriene receptor antagonist. Further, reactive oxygen species generation by PBMCs was induced to a higher degree by patient exosomes compared to healthy exosomes. These findings add to an emerging picture of exosomes as mediators and disseminators of inflammation, and open for further investigations of the link between CCL2 and exosomal leukotrienes in sarcoidosis.

High Levels of miR-483-3p Are Present in Serum Exosomes Upon Infection of Mice With Highly Pathogenic Avian Influenza Virus.

Exosomes, the extracellular vesicles that contain functional proteins and RNAs, regulate cell-cell communication. Recently, our group reported that levels of various microRNAs (miRNAs) in bronchoalveolar lavage fluid exosomes were highly increased in influenza virus-infected mice and that one of those miRNAs, miR-483-3p, was involved in the potentiation of the innate immune responses to influenza virus infection in mouse type II pneumocytes. Here, we evaluated exosomal miR-483-3p levels in the serum of influenza virus-infected mice and found that miR-483-3p levels were significantly increased during infection with a highly pathogenic avian H5N1 influenza virus. Moreover, miR-483-3p-enriched exosomes derived from type II pneumocytes potentiated the expression of proinflammatory cytokine genes in vascular endothelial cells. Our findings suggest that serum exosomal transfer of miR-483-3p might be involved in the inflammatory pathogenesis of H5N1 influenza virus infection.

Upregulation of E‐cadherin in bronchoalveolar lavage fluid‐derived exosomes in patients with lung cancer

BackgroundLung cancer features extremely high rates of morbidity and mortality. Bronchoalveolar lavage fluid (BALF), obtained by bronchoscopy and bronchoalveolar perfusion, can provide information on the cellular components of the lung microenvironment to assist with diagnosis and treatment of lung cancer.MethodsBALF was performed using a flexible bronchofiberscope. Exosomes were collected by ultracentrifugation. ELISA detected the amount of E‐cadherin. Transmission electron microscopic, ELISA and WB were conducted to identify the existence of the exosomes. Transwell and Wound healing assays were used to detect the ability of migration and invasion.ResultsWe identified the existence of exosomes in BALF. Furthermore, we observed larger amounts of E‐cadherin in the BALF obtained from patients with lung cancer than in the control obtained from the healthy side of pneumonia. Exosomes from lung cancer groups promoted the migration and invasion of A549 cancer cells.ConclusionThe exosomes from lung cancer BALF promoted the migration and invasion of A549 cancer cells by carrying E‐cadherin. E‐cadherin on the surface of exosomes may act through a VE‐cadherin dependent mechanism and induce lung cancer metastasis.

Bronchoalveolar Lavage Fluid-Derived Exosomes: A Novel Role Contributing to Lung Cancer Growth.

Exosomes are nanovesicles produced by a number of different cell types and regarded as important mediators of cell-to-cell communication. Although bronchoalveolar lavage fluid (BALF) has been shown to be involved in the development of tumors, its role in lung cancer (LC) remains unclear. In this article, we systemically studied BALF-derived exosomes in LC. C57BL/6 mice were injected with Lewis lung carcinoma cells and exposed to non-typeable Haemophilus influenza (NTHi) lysate. The analysis showed that the growth of lung tumors in these mice was significantly enhanced compared with the control cohort (only exposure to air). Characterization of the exosomes derived from mouse BALF demonstrated elevated levels of tumor necrosis factor alpha and interleukin-6 in mice exposed to NTHi lysates. Furthermore, abnormal BALF-derived exosomes facilitated the development of LC in vitro and in vivo. The internalization of the BALF-derived exosomes contributed to the development of LC tumors. Collectively, our data demonstrated that exosomes in BALF are a key factor involved in the growth and progression of lung cancer.

Diagnostic value of microRNAs derived from exosomes in bronchoalveolar lavage fluid of early-stage lung adenocarcinoma: A pilot study.

BackgroundLow‐dose computed tomography can identify smaller nodules more often than chest radiography in lung screening. However, complications from invasive diagnostic procedures performed to detect nodules are common. Exosomes contain a diverse array of biomolecules that reflect the biological state of the cell from which they are released. The aim of this study was to investigate the diagnostic value of bronchoalveolar lavage (BAL) fluid exosomal microRNAs (miRNAs) for early‐stage lung adenocarcinoma.MethodsWe evaluated miRNAs (miR‐7, miR‐21, miR‐126, Let‐7a, miR‐17, and miR‐19) known to have diagnostic value for lung adenocarcinoma. Exosomes were isolated from the BAL fluid of control subjects (n = 15) and patients with lung adenocarcinoma (n = 13). Exosomal miRNA was analyzed using a commercial kit containing probes targeting six selected miRNAs. Results were validated via quantitative PCR.ResultsThe presence of miRNAs was confirmed in exosomes from BAL fluid of both lung adenocarcinoma patients and control subjects. miR‐126 (P < 0.001) and Let‐7a (P = 0.015) levels were significantly higher in the BAL fluid of lung adenocarcinoma patients than in control subjects. The BAL fluid miRNA signature was confirmed using an independent set of paired adenocarcinoma and normal tissue samples (n = 4). Lung adenocarcinoma tissues showed increased expression of miR‐126 (P = 0.039) compared to normal tissue samples.ConclusionWe identified a close correlation between BAL fluid exosomal miRNAs and tumor miRNAs. BAL fluid exosomal miRNAs obtained through noninvasive methods could serve as diagnostic biomarkers in early‐stage lung adenocarcinoma.

Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses

ABSTRACTBackground: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity.Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing.Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or −80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS.Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at −80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound “peri-exosomal” proteins. In preparations stored at −80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures.Conclusions: Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation.

RNA-containing exosomes in induced sputum of asthmatic patients

RNA-containing exosomes in induced sputum of asthmatic patients

Pulmonary sarcoidosis is associated with exosomal vitamin D–binding protein and inflammatory molecules

Sarcoidosis is an inflammatory granulomatous disorder characterized by accumulation of TH1-type CD4+ Tcells and immune effector cells within affected organs, most frequently the lungs. Exosomes are extracellular vesicles conveying intercellular communication with possible diagnostic and therapeutic applications. We aimed to provide an understanding of the proinflammatory role of bronchoalveolar lavage fluid (BALF) exosomes in patients with sarcoidosis and to find candidates for disease biomarkers. We performed a mass spectrometric proteomics characterization of BALF exosomes from 15 patients with sarcoidosis and 5 healthy control subjects and verified the most interesting results with flow cytometry, ELISA, and Western blot analyses in an additional 39 patients and 22 control subjects. More than 690 proteins were identified in the BALF exosomes, several of which displayed significant upregulation in patients, including inflammation-associated proteins, such as leukotriene A4 hydrolase. Most of the complement-activating factors were upregulated, whereas the complement regulator CD55 was seen less in patients compared with healthy control subjects. In addition, for the first time, we detected vitamin D-binding protein in BALF exosomes, which was more abundant in patients. To evaluate exosome-associated vitamin D-binding protein as a biomarker for sarcoidosis, we investigated plasma exosomes from 23 patients and 11 healthy control subjects and found significantly higher expression in patients. Together, these data contribute to understanding the role of exosomes in lung disease and provide suggestions for highly warranted sarcoidosis biomarkers. Furthermore, the validation of an exosome-associated biomarker in the blood of patients provides novel, and less invasive, opportunities for disease diagnosis.

Altered microRNA profiles in bronchoalveolar lavage fluid exosomes in asthmatic patients

Asthma is characterized by increased airway narrowing in response to nonspecific stimuli. The disorder is influenced by both environmental and genetic factors. Exosomes are nanosized vesicles of endosomal origin released from inflammatory and epithelial cells that have been implicated in asthma. In this study we characterized the microRNA (miRNA) content of exosomes in healthy control subjects and patients with mild intermittent asthma both at unprovoked baseline and in response to environmental challenge. To investigate alterations in bronchoalveolar lavage fluid (BALF) exosomal miRNA profiles due to asthma, and following subway air exposure. Exosomes were isolated from BALF from healthy control subjects (n=10) and patients with mild intermittent asthma (n=10) after subway and control exposures. Exosomal RNA was analyzed by using microarrays containing probes for 894 human miRNAs, and selected findings were validated with quantitative RT-PCR. Results were analyzed by using multivariate modeling. The presence of miRNAs was confirmed in exosomes from BALF of both asthmatic patients and healthy control subjects. Significant differences in BALF exosomal miRNA was detected for 24 miRNAs with a subset of 16 miRNAs, including members of the let-7 and miRNA-200 families, providing robust classification of patients with mild nonsymptomatic asthma from healthy subjects with 72% cross-validated predictive power (Q(2)=0.72). In contrast, subway exposure did not cause any significant alterations in miRNA profiles. These studies demonstrate substantial differences in exosomal miRNA profiles between healthy subjects and patients with unprovoked, mild, stable asthma. These changes might be important in the inflammatory response leading to bronchial hyperresponsiveness and asthma.

Bronchoalveolar lavage fluid exosomes contribute to cytokine and leukotriene production in allergic asthma

Leukotrienes (LTs) are potent pro-inflammatory mediators involved in asthma. Exosomes, nanosized vesicles released from various cells, can stimulate or down-regulate immune responses, depending on the state and nature of the originating cell. We have recently shown an altered exosome profile in bronchoalveolar lavage fluid (BALF) of patients with sarcoidosis, but their role in asthma is unknown. Our aims were to investigate whether exosomes from BALF have LT biosynthetic capacity and to explore phenotypic and functional characteristics of BALF exosomes in asthma. Bronchoalveolar lavage fluid exosomes were collected from healthy individuals (n = 13) and patients with mild allergic asthma to birch pollen (n = 12) before and after birch allergen provocation. Exosomes were characterized by flow cytometry and Western blot. Their capacity to induce IL-8 and LT production in the human bronchial epithelial cell (BEC) line 16HB14o- was measured by ELISA and reverse-phase HPLC, respectively. Compared to BALF exosomes from healthy individuals, BALF exosomes from asthmatics displayed higher levels of exosome-associated markers, such as the tetraspanins CD63 and CD81 and the scavenger receptor CD36. No major differences were observed between BALF exosomes from before and after allergen provocation. Furthermore, we show that BALF exosomes contain enzymes for LT biosynthesis. The effect of exosomes to promote LTC(4) and IL-8 release in BEC was significantly increased for exosomes from asthmatics, and the CysLT(1) receptor antagonist Montelukast reduced exosome-induced IL-8 secretion. Bronchoalveolar lavage fluid exosomes from asthmatic and healthy individuals exhibit distinct phenotypes and functions. BALF exosomes from asthmatics might contribute to subclinical inflammation by increasing cytokine and LTC(4) generation in airway epithelium.

Proinflammatory exosomes in bronchoalveolar lavage fluid of patients with sarcoidosis

BackgroundSarcoidosis is a systemic disease of unknown aetiology characterised by granuloma formation and the presence of interferon γ (IFNγ)-producing T cells that cause inflammation and tissue damage in multiple organs,...

Exosomes with major histocompatibility complex class II and co-stimulatory molecules are present in human BAL fluid.

Exosomes are 30-100 nm diameter vesicles formed by inward budding of endosomal compartments and are produced by several cell types, including T-cells, B-cells and dendritic cells (DC)s. Exosomes from DCs express major histocompatibility complexes (MHC) class I and II, and co-stimulatory molecules on their surface, and can induce antigen-specific activation of T-cells. The aims of the present study were to investigate for the presence of exosomes in bronchoalveolar lavage fluid (BALF) from healthy individuals, and to establish if these exosomes bear MHC and co-stimulatory molecules. The authors analysed BALF taken from seven healthy volunteers and used exosomes from monocyte-derived DC (MDDC) cultures as a reference. After ultracentrifugation, exosomes were bound to anti-MHC class II coated magnetic beads and analysed by flow cytometry and electron microscopy. The authors report for the first time that exosomes are present in BALF. These exosomes are similar to MDDC derived exosomes as they express MHC class I and II, CD54, CD63 and the co-stimulatory molecule CD86. The results demonstrate that exosomes are present in the lung, and since they contain both major histocompatibility complex and co-stimulatory molecules it is likely that they are derived from antigen presenting cells and might have a regulatory role in local immune defence.