Introduction

Respiratory disorders encompass a range of acute and chronic disorders caused by disordered phenotypes at the cellular, tissue levels. Chronic respiratory diseases have emerged as the third most prevalent disease worldwide, with a global prevalence of approximately 7.13%, which accounted for approximately 7.00% of all global deaths and caused a total of 112.32 million disability-adjusted life-years.1 However, the heterogeneity and complexity of respiratory diseases give rise to clinical practice challenges, including the unclarity of aetiology, insufficiency of specific biomarkers and the limitation of targeted therapeutic agent.

Recent studies have demonstrated the development of respiratory disorders entailed a convoluted and multistage evolutionary process arising from the intricate interplay of genetic/epigenetic factors and environmental factors.2 3 Among these factors, exposure to environmental pollutants has emerged as a substantial contributor to the induction and exacerbation of respiratory diseases, garnering considerable attention due to their controllable and preventable characteristics.4 Environmental pollutants encompass substances that directly or indirectly jeopardise human health, and perturb the composition and characteristics of the natural ecological environment, which can be classified into chemical (eg, PM2.5, cigarette smoke and ozone), biological (eg, bacterial, viral and parasitic infections) and physical (eg, radiation, cold and light pollution) pollutants based on their nature and origin.5 Elucidating the mechanism of respiratory diseases induced by environmental pollutants is of great significance for early prevention, diagnosis, disease course monitoring and treatment development in clinic.

Extracellular vesicles (EVs), which act as pivotal carriers of emerging cellular communication, have been shown to play a crucial role in the pathological processes underlying environmental pollutants-induced respiratory disorders.6 7 Environmental pollutant exposure has been revealed to stimulate airway barrier cells and micro-organism to promote the secretion of EVs carrying disordered contents, which influence the biological functions of disease-effector cells, ultimately contributing to the development of respiratory disorders, including respiratory dysregulation and respiratory diseases.4 8 Moreover, EVs possess significant potential as biomarkers and therapeutics for respiratory disorders due to their unique stability and disease specificity. In this review, we reviewed and summarised the regulatory mechanism of environmental pollutants exposure-derived EVs (EPE-EVs) in respiratory disorders, and their potential for clinical transformation.

Regulatory mechanisms of EPE-EVs in respiratory disorders

Disordered intercellular communication is regarded to be involved in early-stage and end-stage respiratory disorders.9 10 EVs secreted by secretory cells under exposure to different physical, chemical and biological environmental pollutants are involved in the regulation of biological function of recipient cells to develop various pathogenic disorders at the cellular and tissue level, such as inflammation, infection, lung dysfunction and even tumour (figure 1 and table 1), consequently contributing to the induction and progression of respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), emphysema, pulmonary fibrosis, lung cancer, pulmonary hypertension and infectious lung diseases (figure 2).

Figure 1

The regulatory mechanisms of EPE-EVs in respiratory disorders. Exposure to physical, chemical and biological pollutants stimulates airway barrier cells (eg, epithelial cells, macrophages) and airway bacteria to release abnormal EVs, that regulate the biological function of effector cells and promote disordered phenotypes by delivering key cargoes including proteins, RNA and toxins (created with Biorender.com). COPD, chronic obstructive pulmonary disease; EVs, extracellular vesicles; EPE-EVs, environmental pollutants derived extracellular vesicles; HDM, house dust mite; LPS, lipopolysaccharides; VEGFA, vascular endothelial growth factor A.

Table 1

The role of EPE-EVs mediating intercellular communication in the respiratory disorders

Figure 2

EPE-EVs as potential biomarkers for respiratory diseases. The sources and contents of EPE-EVs possessing biomarker potential for various respiratory diseases are sorted out. Different sections of the ring represent different respiratory diseases. The assorted colors of the fonts in the outer boxes represent diverse environmental pollutants derivation of EVs contents. EVs, extracellular vesicles; EPE-EVs, environmental pollutants derived extracellular vesicles; HDM, house dust mite; LPS, lipopolysaccharides; PM, particulate matter; VEGFA, vascular endothelial growth factor A.

Chemical pollutants exposure derived EVs in respiratory disordersCigarette smoke exposure

Cigarette smoke contains various airway cytotoxic components, including known toxins, carcinogens, oxides and solid particles, among others.11 These toxic components promoted airway inflammation, disrupted the epithelial barrier, vascular endothelium and fibroblast functions, and further caused pathological changes mainly characterised by airway remodelling, lung extracellular matrix and tissue damage, which consequently contributed to the progression of diseases such as COPD, emphysema, lung cancer, pulmonary hypertension and pulmonary fibrosis, among others.

Airway inflammation: Airway inflammation is the underlying pathological changes in respiratory diseases, like COPD and asthma.12 Héliot et al have reported that smoking caused significant downregulation of let-7e, let-7g, miR-26b in EVs in human BAL (bronchoalveolar lavage fluid), which were internalised by human bronchial epithelial cells to significantly upregulated the expression of IL-6, thus promoting airway inflammation.13 Khodayari et al have found that cigarette smoke exposure stimulated human small airway epithelial cells to release abnormal EVs, which induced increased expression and release of GM-CSF and IL-8 in AATD (single-nucleotide mutation SERPINA1 gene) macrophages through ER stress and NF-κB pathways, thereby promoting neutrophilic inflammation formation.14 Since EVs secreted by dendritic cells have been shown to promote CD4+T cell activation,15 Russell et al have identified that cigarette smoke extract (CSE) exposure significantly increased the release of EVs from human dendritic cells, which promoted CD4+T cells differentiation towards Th17 or Th1 phenotypes through dose-dependently upregulating the levels of RORγt, FoxP3 and IL-17A,16 suggesting a tendency to promote allergic asthma,17 but the key contents and specific mechanisms of EVs remain to be further explored. It is important to note that the types of cigarette smoke exposure (smoking, secondhand smoke and third-hand smoke) and dosage should be considered due to their tendency to induce different types of inflammation.18 19 In addition, the direction of M0 macrophage polarisation has been demonstrated to mediate the direction of airway inflammation, and the M1 polarisation type has been found as one of the mechanisms of COPD aggravation.20 21 Wang et al have revealed that CSE stimulated abnormal EVs secreted by mouse aortic endothelial cells in COPD mice, which were internalised by mouse macrophages and promoted M1 polarisation by upregulating TREM-1 expression, thus leading to exacerbation of COPD.22

Epithelial barrier dysfunction: Epithelial cell death and destruction of the extracellular matrix are crucial histological lesions in diseases such as emphysema in COPD.23 24 Moon et al have reported that CSE stimulated the secretion of flCCN1, cCCN1 and plasmin-enriched EVs by lung epithelial cells, which were internalised by adjacent and distant lung epithelial cells, thus activating the Rho pathway by upregulating MMP-1 through the interaction between cCCN1 and integrin-7, promoting the epithelial cell death and tissue loss, subsequently causing the deterioration of emphysema in COPD patients.25 Li et al have identified that CSE exposure-induced human macrophages to produce EVs containing high levels of MMP14 by activating the JNK/MAPK signalling pathway and promoting apoptosis, which mediated the destruction of airway extracellular matrix.26 Also, Xia et al have observed that CSE exposure caused the secretion of upregulated miR-93 in EVs by human bronchial epithelial cells through upregulation of METTL3 expression, which entered human macrophages and activated the JNK signalling pathway by targeting DUSP2, elevating MMP9 and MMP12 levels, consequently inducing elastin degradation and emphysema.27 Compared with the negative control, smoking increased the level of miR-93 and the degree of elastin degradation in the serum of patients with COPD more significantly than that of smokers only,27 supporting the evidence that smoking-induced emphysema complication of COPD.28 29

Vascular dysfunction: The abnormal proliferation and migration of pulmonary vascular smooth muscle cells is the basis of vascular remodelling in pathological pulmonary hypertension.30 Saxena et al have identified that CSE induced the secretion of vascular endothelial growth factor A (VEGFA) in EVs from human airway basal cells, which promoted abnormal proliferation of vascular endothelial cells through the VEGFR2 signalling pathway, thus contributing to the vascular endothelial dysfunction.31 Zhu et al have discovered that CSE stimulated the secretion of spermine-enriched EVs by mouse endothelial cells, which lead to abnormal contraction and proliferation of mouse pulmonary artery smooth muscle cells by activating the spermine/CaSR signalling pathway, thus promoting the progression of pulmonary hypertension.32 Su et al have also detected that CSE-induced mouse endothelial cells to secrete upregulated miR-1249 into EVs, which contributed to the high proliferation of mouse pulmonary artery smooth muscle cells and the formation of an antiapoptotic state by inhibiting the HDAC10-NFκB-CaSR cascade, thus accelerating the development of pulmonary hypertension.33

Lung fibroblasts dysfunction: Abnormal differentiation of lung fibroblasts has been found to involved in the airway remodelling, thus contributing to the development of COPD and pulmonary fibrosis.34 Xu et al have reported that CSE stimulated human bronchial epithelial cells to secrete upregulated miR-21 into EVs, which induced differentiation towards myofibroblasts of human embryonic lung fibroblasts through the activation of pVHL/HIF-1α signalling pathway and upregulation of targeted gene α-SMA, thereby inducing airway remodelling and COPD.35 Bai et al have found that CSE exposure specifically downregulated the expression level of circRNA_0026344 in human bronchial epithelial cells to select and released high levels of miR-21 into EVs, which were internalised by human lung fibroblasts and targeted to silence Smad7 gene, activated TGF-β1/Smad3 pathway, consequently mediating fibroblast differentiation and extracellular matrix accumulation, thereby exacerbating abnormal epithelial-fibroblast crosstalk in pulmonary fibrosis.36

Tumourigenesis: The association between cigarette smoke exposure and lung cancer has been widely established.37 Cheng et al have found that CSE-induced human M2 tumour-associated macrophages to release EVs with high levels of circEML4, which were internalised by human lung squamous cell and reduced the distribution of ALKBH5 in the nucleus through interaction with ALKBH5, resulting in increased m6A modification of SOCS2 gene, causing the activation of JAK-STAT signalling pathway and enhancement of the proliferation and invasion of non-small cell lung cancer cells.38 Malyla et al have also revealed that CSE exposure stimulated human bronchial epithelial cells to secrete EVs containing high levels of β-catenin mRNA and was internalised by other healthy bronchial epithelial cells, inducing tumourigenesis by activating the WNT/β-catenin signalling pathway.39

Particulate matter exposure

Particulate matter (PM) is a variety of solid or liquid particles evenly dispersed in the atmosphere of different diameters, which adsorb various toxic substances, deposit and damage different locations of the airway.40 As an exogenous airway toxicant, PM is involved in lung injury and fibrosis through promoting inflammation and distorting macrophage function, as well as damaging epithelial barrier and airway smooth muscle function, causing asthma and even promoting lung cancer.

Airway inflammation: The polarisation of macrophages and secretion of inflammatory factors are one of the important pathological processes of diseases such as COPD and asthma.41 42 Martin et al have found that PM2.5-0.3 exposure stimulated abnormal EVs secretion from human macrophages, which caused significant cytotoxicity and upregulation of the expression levels of IL-8, IL-6 and TNF-α in bronchial epithelial cells.43 Shin et al have also reported that PM stimulated human nasal epithelial cells to secrete EVs containing significantly upregulated miR-19a and miR-614, which were targeted to induce M0 macrophages to M1 polarisation by downregulating the expression of RORα, and promoted the secretion of inflammatory factors including iNOS and TNF.44

Lung injury: The process from acute alveolar injury to fibrotic repair is the main pathological evolution of lung injury.45 Hu et al have revealed that PM2.5 exposure stimulated mouse macrophages to release EVs containing high levels of TNF-α, which were internalised by mouse lung epithelial cells and upregulated surfactant protein expression via activating of thyroid transcription factor-1, thereby mediating PM2.5-induced pneumonoedema in acute lung injury.46 Park et al have found that PM10 and PM2.5 exposure stimulated the production of EVs containing high levels of miR-6238 in human lung epithelial cells, which were endogenous to alveolar macrophages, inhibited the expression of CXCL3 and alleviated neutrophil chemotaxis, thereby regulating the transition of PM-induced acute lung injury from the injury phase to the fibrotic phase.47

Airway hyperresponsiveness and remodelling: Airway hyperresponsiveness and airway remodelling are the two most important end-effect pathological changes in asthma.48 Our previous study has reported that PM2.5 exposure stimulated bronchial epithelial cells to secrete significantly upregulated let-7i-5p in EVs, which entered normal human bronchial epithelial cells ‘horizontally’ or human sensitive smooth muscle cells ‘vertically’, inhibited the expression of target gene DUSP1, and activated the MAPK signalling pathway by increasing the phosphorylation levels of PI3K, AKT and p38, consequently causing the disruption of epithelial barrier defenses and enhancement of the contractile ability of smooth muscle cells, leading to childhood asthma.49 Mechanically, oxidative stress and DNA damage have been identified to be one of the main cytotoxic effects of PM2.5.50 51 Zheng et al have also demonstrated that PM2.5 stimulated the high release of EVs lncRNA PAET from bronchial epithelial cells, which disrupted oxidative phosphorylation metabolism, decreased adenosine triphosphate (ATP) production and reactive oxygen species (ROS) accumulation, as well as increased DNA damage in recipient epithelial cells by both enhancing the stability and accumulation of METTL3 and stimulating the modification and reduction of COX4I1 level in a YTHDF3-dependent manner, consequently mediating airway remodelling and inducing childhood asthma.52 This study extended the understanding of the toxicology and identified a potential target for childhood asthma.

Tumourigenesis: PM has been suggested to be associated with an increased risk of lung cancer, but the exact mechanism remains to be explored.53 Xu et al have reported that PM2.5 exposure stimulated adenocarcinoma cells to release upregulated Wnt3a encapsulated in EVs, which entered other adenocarcinoma cells and promoted their invasion and abnormal cell proliferation by activating the Wnt3a/β-catenin signalling pathway.54

Ozone exposure

Ozone, a small molecule of strongly irritating gas of low solubility that can cross the respiratory barrier, directly causes acute inflammation and respiratory cytotoxicity.55 Carnino et al have investigated that ozone-stimulated mouse CD45+ myeloid cells to release significantly upregulated miR-21 and miR-145 and downregulated miR-24-3 p and miR-20 in EVs, which were targeted into mouse macrophages, induced the synthesis and release of iNOS, CXCL-1, CXCL-2 and IL-1b, further promoting the formation and aggravation of airway inflammation.56

Biological environmental pollutants derived EVs in respiratory disordersBacteria exposure

Bacterial exposure derived EVs are involved in the development of infectious lung diseases and respiratory system injury in two pathways: one is through autocrine EVs that encapsulate the bacterial wall components and toxins (called outer membrane vesicles)57 to induce neutrophil inflammation and vascular injury; and the other is bacterial to stimulate the production of disordered EVs by the infected cells,58 to promote the release of inflammatory factors and immune evasion.59

Neutrophil inflammation: Neutrophil inflammation is a common and important innate immune response of the body against a variety of pathogens, which is related to the progression of pathological changes such as lung injury and emphysema in COPD.60 Bacterial infections have been reported as the second leading cause of death worldwide, with 950 000 deaths caused by lower respiratory tract infections associated with Escherichia coli.61 Lee et al have identified that EVs released by Escherichia coli were internalised by human microvascular endothelial cells, which upregulated the expression and release of IL-8 and CXCL1 in endothelial cells by activating the TLR4/NF-κB signalling pathway, thus promoting neutrophil recruitment and neutrophil inflammation formation.62 Kim et al have also identified that EVs secreted by E. coli were internalised by alveolar type II cells and induced elevated levels of IL-17A release and neutrophil inflammation by activating the TLR4 signalling pathway, consequently leading to the development of emphysema as complications of COPD.63 In addition to the conventional pathogens, the control of tuberculosis remains a worldwide problem of infectious lung disease.64 Singh et al have demonstrated that EVs released by mouse macrophages infected with Mycobacterium tuberculosis were interiorised by other uninfected macrophages, upregulating the expression and release of their chemokines RANTES and MIP-1α, and promoting the recruitment and enrichment of macrophages and neutrophils.65 Notably, neutrophil inflammation has been found to be involved in driving Th1/Th17 cell-mediated adaptive immunity.66 Kim et al have reported that Gram-negative bacteria secreted lipopolysaccharides (LPS) enriched EVs, which were internalised by airway epithelial cells and alveolar macrophages, inducing neutrophilic inflammation and infiltration of Th1 and Th17 cells.67 In addition, Meganathan et al have found that bacterial EVs extracted from organic dust in chicken farms significantly upregulated the expression of IL-8, IL-6 and TNF-α, etc in lung epithelial cells by activating the NF-κB signalling pathway, to induce neutrophilic inflammation formation, subsequently promoting remodelling injury of the lung including collagen deposition and epithelial thickening.68

Release of inflammatory cytokine: Bacterial-derived EVs promote the release of chemokines, interleukins and tumour necrosis factor by stimulating macrophage-dominated airway barrier cells in a toll-like receptor-dependent manner. Marion et al have reported that intranasal exposure to EVs produced by Acinetobacter baumannii-induced upregulation of CCL2, TNF-α and IFN-γ levels in mouse BAL in a TLR2 and TLR4 dependent manner.69 Athman et al have found that mouse macrophages infected with M. tuberculosis released EVs containing liposaccharides and lipoglycoproteins, which were internalised by uninfected macrophages and upregulated the synthesis and release of IL-8 and TNF-α through activation of TLR2 signalling pathway.70 Mehanny et al have identified that EVs released by Streptococcus pneumoniae were internalised by human lung epithelial cells, keratinocytes and macrophages, which upregulated the expression and release of TNF-α in dendritic cells.71 Kovach et al have found that both heat-killed and live Klebsiella pneumoniae and S. pneumoniae stimulated human and mouse macrophages to release EVs containing high levels of IL-36γ via TLR3, thereby modulating the host inflammatory response.72 Interestingly, Jung et al have found that in the process of Legionella pneumophila infection, disordered EVs derived from infected human macrophages were internalised by uninfected epithelial cells, upregulating the release and expression of IL-6 and CCL2, while viable bacteria component in EVs derived from L. pneumophila were internalised by uninfected macrophages via TLR2, upregulating the release and expression of TNF-α, IL-1β and CCL2, suggesting differences in the reactivity of airway cells to different components of bacteria-derived EVs.73

Immune evasion: The release of exogenous EVs to impair host immune function has been revealed to be a strategy for bacterial invasion of the respiratory system.74 Codemo et al have identified that EVs produced by S. pneumoniae containing pulmonary haemolysin and pneumococcal surface protein C not only were internalised by human lung epithelial cells and human monocyte derived dendritic cells, upregulating the release and expression of IL-6, IL-8, IL-10 and TNF in dendritic cells but also exposed the target of complement C3b deposition and membrane attack complex formation in serum, and impaired phagocytosis activity of macrophages, consequently evaded host immune response.75 Armstrong et al have revealed that EVs released by Pseudomonas aeruginosa were internalised by human lung macrophages and significantly downregulated the gene and protein expression levels of 11 MHC Class I and Class II related molecules, suggesting that EVs released by P. aeruginosa mediated immune evasion.76

Vascular dysfunction: Bacterial-derived EVs also mediates pulmonary vascular endothelial injury induced by severe bacterial infection sepsis. Laakmann et al have illustrated that LPS containing EVs released by E. coli, K. pneumoniae and Salmonella enterica could be internalised by human microvascular lung endothelial cells, which inhibited the expression level of RNase1 in endothelial cells through LPS-dependently activation of the TLR4/MyD88/IRAK-1/p38 pathway, thereby promoting the development of vascular endothelial dysfunction and lung injury.77

Virus exposure

The infection of common respiratory viruses (rhinovirus, influenza virus, etc) is one of the crucial causes of infectious lung diseases.78 Promoting airway inflammation, immune evasion and bacterial coinfection are important mechanism of virus-derived EVs promoting the aggravation of infectious lung diseases.

Airway inflammation: Respiratory viruses promote airway inflammation mainly by infecting epithelial cells and distorting intercellular communication with inflammatory cells such as macrophages. Mills et al have found that rhinovirus infection stimulated airway epithelial cells to release Tenascin-C-rich EVs by activating the TLR3 pathway, which is internalised by uninfected epithelial cells and up-regulated the expression and release of CXCL8, IL-6 and CCL5, thus enhancing airway inflammation.79 Xia et al have reported that influenza A virus infection stimulated abnormal EVs secretion from lung epithelial cells, which were internalised by uninfected macrophages, promoting their M1 polarisation and expression levels of iNOS, TNF-α and IL-1β.80 Zhu et al have also revealed that influenza A virus infection stimulated mouse lung epithelial cells to release EVs containing high levels of miR-1249-5 p, which were internalised by mouse macrophages and inhibited NF-kB signalling pathway by down-regulating the expression of SLC4A1, consequently promoting the expression of IL-6 and TNF-α.81

Immune invasion: Viruses stimulate infected host cells to release abnormal endogenous EVs that target uninfected cells, thus contributing to the impairment of anti-infective function. Xia et al have reported that SARS-CoV-2 infection stimulated human alveolar epithelial cells and human lung adenocarcinoma cells to release 1.6–9.5 µm diameter EVs containing a large number of viral particles, which were internalised by uninfected human alveolar epithelial cells, thereby transmitting SARS-CoV-2 virus resistance to neutralising antibodies.82 Berry et al have found that SARS-CoV-2 infection stimulated human nasal epithelial cells to secret EVs containing ACE2 (SARS-CoV-2 receptor) and TMPRSS2 (protease allowing SARS-CoV-2 to invade cells) into nasal mucus and were internalised by uninfected nasal epithelial cells, thus promoting virus infection through TMPRSS2-dependent means.83

Promoting bacteria coinfection: Recent studies have demonstrated that respiratory viruses can promote bacterial coinfection through various mechanisms, such as impairing host immunity, remodelling tissues, enhancing bacterial adhesion, and expressing surface receptors and adhesion proteins.84 85 And syntrophic effect has been revealed as a new mechanism.86 Hendricks et al have reported that RSV infection increased the host iron-binding proteins on the EVs surface of human bronchial epithelial cells, which interacted with P. aeruginosa biofilm to transfer nutrient iron and promote the growth of bacterial biofilm.87

Fungus exposure

Fungal lung disease (Aspergillus, Candida, Pneumospora and Cryptococcus, etc) is often one of the end-stage complications of immune dysfunction and severe chronic lung disease.88 Considering elucidating the mechanism of changes in virulence traits of fungi is one of the important problems in antifungal infection,89 Octaviano et al have reported that EVs released by Paracoccidioides brasiliensis recovered the virulence traits of the virulent variant attenuated strain, and upregulated the secretion levels of TNF-α, IL-6 and NO in macrophages, thus exacerbating infection in mice.90

House dust mite exposure

The association between house dust mite (HDM) exposure and allergic respiratory diseases such as asthma and sinusitis has been widely discussed.91 92 Zhang et al have reported that HDM stimulated human bronchial epithelial cells to release specific EVs containing HDM components, which activated the Notch2 signalling pathway of dendritic cells through the CNTN1 receptor on the EVs membrane surface, to promote the differentiation process of CD4+ T cells to Th2/Th17 cells mediated by CD40 and MHC class II receptors of activated dendritic cells, consequently inducing allergic asthma.93

Physical pollutants exposure derived EVs in respiratory disorders

It is noteworthy that EPE-EVs helped elucidate the mechanism radiation affecting intercellular communication between cancer cells and immune microenvironment, which is an important issue for the improvement of tumour therapy.94 95 For lung cancer, Zheng et al have reported that EVs containing upregulated miR-23a secreted by lung adenocarcinoma cells stimulated by radiation inhibited the expression of PTEN in human umbilical vein endothelial cells, upregulated the phosphorylation of AKT, enhanced its proliferation and migration, thus promoting the formation of tumour blood vessels.96

Previously, it has been hypothesised that the impact of seasonal changes on virus infection activity is associated with viral activity and modes of transmission.97 98 Huang et al have found that cold exposure at 32℃ reduced the abundance of miR-17, and surface receptor proteins including low-density lipoprotein receptor (LDLR) and intercellular adhesion molecule 1 (ICAM-1) in EVs secreted by rhinovirus-infected inferior human nasal epithelial cells, thereby impairing the antiviral activity mediated by the internalisation of EV by other nasal epithelial cells and the decoy action of EV surface receptor proteins.99

The potential and applications of EPE-EVs in clinical respiratory medicineEPE-EVs as biomarkers in respiratory diseases

Compared with traditional biomarkers, EPE-EVs possess several advantages: (1) Stability. Compared with traditional biomarkers that may have the disadvantage of decomposability, the lipid membrane structure of EVs has good stability under in vivo and in vitro preservation conditions.100 101 (2) Specificity. The specific contents of EPE-EVs have been shown to correlate with levels of exposure to environmental pollutants.49 102 In addition, as mentioned above, EPE-EVs have the specificity to reflect the phenotype of abnormal cell tissue in the disease state, which helps to reveal the detailed classification of the disease. (3) Convenience. Currently, clinical diagnosis methods to assess EVs in of respiratory diseases (eg, lung tissue biopsy, bronchial biopsy and BAL) via bronchoscopy are limited due to the procedure invasiveness. While EVs have been stably isolated and detected in a variety of non-invasive samples (plasma, urine and exhaled breath, etc).103 104 (4) Sensibility. Distinguishing between disease signals and background noise is a great challenge in extraction of traditional free biomarker.105 106 However, EVs contents have been found to increase the sensitivity of genetic mutation detection (eg, lung cancer and disease identification).107 108

EVs as potential biomarkers in respiratory diagnosis and disease course monitoring have been preliminarily explored (table 2). First, EPE-EVs have shown efficacy in the diagnosis of respiratory diseases and infection. For childhood asthma, our previous study has revealed that PM2.5 altered the plasma EVs miRNA profile, discovered that let-7i-5p in EVs with upregulated expression as biomarkers and displayed a higher area under the curve (AUC=0.86) when combining serum IgE than when only using let-7i-5p in EVs (AUC=0.79).49 A series of plasma EVs contents have been screened for diagnosis of M. tuberculosis infection, including miR-484, miR-425 and miR-96 combined (AUC=0.78), ASdes and MTB-miR5 combined (AUC=0.99).109 110 Elucidating the disordered EVs multiomics profiles of airway microbiome is a new direction of biomarker study.111 A diagnostic model for asthma, COPD and lung cancer has been established by Yang et al using antimicrobial EVs antibody titre analysis based on the detection of antibodies targeted to key bacteria-secreted EVs in serum samples, while adding smoking status as a covariate increased the AUC from 0.73 to 0.78 for asthma, 0.64 to 0.79 for COPD, and 0.74 to 0.80 for lung cancer.112 The composition and functional characteristics of bacterial microbial-derived EVs in the urine of children with allergic airway disease have been identified by Samra et al, and further validation of their diagnostic efficacy in the population is required.113 Additionally, EPE-EVs may help provide auxiliary information on the monitoring of disease severity or infection status. Based on naive Bayesian machine learning model, Meng et al have identified hsa_circ_0005045 in plasma EVs as a biomarker of PM2.5-induced COPD exacerbation (AUC=0.77).114 Fujita et al have revealed that the COPB2 protein in plasma EVs predicted the severity of COVID-19 (AUC=0.85).115

Table 2

EPE-EVs as potential biomarkers of respiratory diseases in diagnosis and prognosis

EPE-EVs as biological agents in respiratory diseases

Micro-organisms and protozoa derived EVs hold promise in the prevention and treatment of respiratory diseases. First, extracting EVs released by pathogens and engineering EPE-EVs are expected to contribute to the development of novel vaccines. Choi et al have found that inoculation with S. aureus derived EVs induced antibody production and a Th1 cell immune response, thus having a protective effect against pneumonia induced by S. aureus infection.116 Jiang et al found that Spike receptor binding domain (RBD) modified EVs derived from Salmonella typhimurium-induced high titre blood anti-RBD IgG and mucosal immunity through intranasal inoculation, thus preventing infection with wild type and Delta variant SARS-CoV-2.117

In addition, pathogenic microbial such as bacteria, virus and protozoa derived EPE-EVs also has the potential as new biotherapeutic agents. Based on the viewpoint that bacteria enhance antiviral function by modulating host immune response, Bierwagen et al have reported that EV released by Klebsiella, E. coli and Salmonella significantly upregulated the expression and release of CXCL8, IL-1b and IL-12b in macrophages and enhanced the antiviral immune function of macrophages, thereby blocking the replication of virus in macrophages.118 Díaz-Godínez et al have also demonstrated that EVs containing ROS released by Entamoeba histolytica Schaudinn in histolytic tissues were internalised by human neutrophils, inhibiting neutrophil respiratory burst and NETosis, suggesting its potential as a therapeutic agent for inflammation.119 In the emerging field of tumour biotherapy, the role of bacteria-derived EVs as antitumour immune activator has been widely discussed.120 121 For medical protozoa, Yang et al have found that plasmodia infected mouse plasma EVs contained high levels of miR-16/322/497/17, which were internalised by vascular mouse endothelial cells in tumour microenvironment, significantly reducing their VEGFR2 expression and migration capacity, thereby inhibiting tumour angiogenesis and growth in Lewis lung cancer.122

Prospect and suggestions of EPE-EVs in clinical respiratory medicine

It is noteworthy that EPE-EVs still have a broad research prospect in mediating the involvement of environmental pollutants in respiratory diseases. However, to overcome the limitations of EPE-EVs in clinical application, several problems and suggestions that needs urgent attention are listed below.

Emerging environmental pollutants: The mechanism of respiratory disorders induced by an emerging type of environmental pollutants including persistent organic pollutants, endocrine disruptors, antibiotics and microplastics, which possess the characteristics of serious harm, environmental persistence, bioaccumulation and complexity of treatment needs urgent attention.123–126 In addition, some emergingly discovered forms of traditional pollutants may have unrecognised potential hazards, such as thirdhand smoke.127

Indoor exposures of outdoor pollutants: Most present studies on outdoor pollutant exposure (PM, volatile organic compounds, etc) are based on outdoor exposure conditions monitored centrally, while there is still a lack of consensus on the composition, nature and concentration changes of indoor exposure of these pollutants.128 129

Biomarkers in clinical screening and prevention: Currently, EPE-EVs have been mainly validated as disease effect biomarkers, but their unique potential to monitor hazardous environmental exposures and predict early-stage respiratory diseases remains largely unexplored. Estimating individual exposure levels to specific environmental pollutants and the risk of developing certain respiratory diseases (such as asthma, COPD and lung cancer) through the detection of specific EPE-EVs is promising. With advances in high-throughput sequencing and multiomics, a combination of traditional screening methods and emerging technologies such as artificial intelligence, machine learning and neural networks is necessary.130 131

EVs-based liquid biopsy: Although a variety of EVs isolation and purification methods have been developed, but their complexity has limited the clinical application.132 Simplified dichotomic size-exclusion chromatography is expected to be clinically adopted in a relatively short time and without relying on large laboratory instruments.133 It is noteworthy that with the deepening understanding of EVs membrane proteins as biomarkers, colloidal gold nanoparticle technology holds the potential to facilitate the development of convenient clinical assay kits.134

Biotherapeutic agents: As previously stated, the potential of microbial and protozoan-derived EVs for combating infections, modulating immunity and antitumour effects has been mentioned. Given the probability that specific microbial exposures protect humans from multiple respiratory diseases, screening of specific microbial species as well as EVs contents, exposure dosage and duration that have a protective effect on respiratory diseases, may contribute to the development of novel biotherapeutic agents.135–137

In general, the prospect of EPE-EVs in clinical respiratory medicine is promising. Given the preventability of environmental pollutants and their crucial role in the pathogenesis of respiratory disorders, further exploration of EPE-EVs can help uncover the toxic mechanisms of pollutants exposure in respiratory dysfunctions and disease development, provide a new perspective for screening pollutants exposure, early diagnosis, developing novel treatment strategies, assessing stage and prognosis in respiratory disorders.

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