The question of this systematic review was to assess whether exosomes could have a potential role in asthma and COPD exacerbations.

The protocol has been submitted to the international prospective register of systematic reviews (PROSPERO registration code: CRD42023483307) and performed in agreement with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) (Moher et al., 2015), with the relative flow diagram reported in Figure 1. This study satisfied all the recommended items reported by the PRISMA 2020 checklist (Page et al., 2021).

The PEO (Population, Exposure, and Outcome) framework was applied to develop the literature search strategy and question, as previously reported (Moola et al., 2015). The “Population” included asthma and COPD; the “Exposure” regarded asthma and COPD exacerbations; the assessed “Outcome” was exosomes.

A comprehensive literature search was performed for research studies, written in English, and investigating the role of exosomes in exacerbations of asthma and COPD. The search was performed in MEDLINE, Scopus, and ClinicalTrials.gov, to identify relevant studies available with no time limit up to 6 September 2023.

The string used for the search in MEDLINE, Scopus, and ClinicalTrials.gov was as follows: “exosomes AND (COPD OR asthma) AND exacerbations”.

Literature search results were uploaded to Eppi-Reviewer 4 (EPPI-Centre Software. London, United Kingdom), a web-based software program for managing and analysing data in literature reviews that facilitates collaboration among reviewers during the study selection process.

Research studies reporting results concerning the role of exosomes in asthma and COPD exacerbations were included in the systematic review.

Two reviewers (L.C. and R.L.) independently checked the relevant studies identified from MEDLINE, Scopus, and ClinicalTrials.gov. The studies were selected in agreement with previously mentioned criteria, and any difference in opinion about eligibility was resolved by consensus.

Data from included studies were extracted and checked for study year and references, PMID or ClinicalTrials.gov identifier, study and subjects’ characteristics, outcomes, main results, and study quality assessment via the Jadad Score (Jadad et al., 1996).

The endpoint of this systematic review was to evaluate the potential role of exosomes in asthma and COPD exacerbations.

Data from original papers were extracted and reported via qualitative synthesis. Statistical significance was identified for p < 0.05.

The risk of bias for included clinical studies was analyzed via the Jadad score (Jadad et al., 1996). The Jadad score, with a scale of 1–5 (score of 5 being the best quality), was used to assess the quality of the papers concerning the likelihood of bias related with randomisation, double blinding, withdrawals, and dropouts (Jadad et al., 1996). Studies were considered of low quality at Jadad score <3, of medium quality at Jadad score = 3, and of high quality at Jadad score >3.

Two reviewers (L.C. and R.L.) independently assessed the quality of studies, and any difference in opinion about the quality score was resolved by consensus.

Of the 24 potentially relevant records screened in MEDLINE, Scopus, and ClinicalTrials.gov, 9 studies were deemed eligible for a qualitative analysis. This systematic review included data obtained from studies investigating the role of exosomes in asthma and COPD exacerbations.

Seven studies (Tan et al., 2017; Yu et al., 2021; Torregrosa Paredes et al., 2012; Meng et al., 2022; Jiang et al., 2023; Song et al., 2020; Wang et al., 2022) were published in full text articles, and 2 studies (ClinicalTrials.gov, 2023b; ClinicalTrials.gov, 2023a) were available only on ClinicalTrials.gov. For 3 of the included studies (Tan et al., 2017; Torregrosa Paredes et al., 2012; Wang et al., 2022) Jadad score was suitable for quality assessment. All were considered of low quality (Jadad score = 0). The main characteristics of the studies included in the systematic review are summarized in Table 1.

In 2017, a study conducted by Tan et al. (Tan et al., 2017) described a correlation between the level of circulating exosomes and the levels of plasma biomarkers of systemic inflammation in COPD patients. Circulating exosomes, identified as CD9⁺ macrovesicles, and plasmatic systemic inflammation biomarkers, such as C-reactive protein (CRP), soluble tumor necrosis factor receptor-1 (sTNFR1) and interleukin (IL)-6, were quantified in patients with acute exacerbation of COPD (n = 20) or stable COPD (n = 20), and non-smoking healthy controls (n = 20). Plasma exosomes were significantly higher in patients with exacerbations (p < 0.001) and stable COPD patients (p < 0.05) compared to healthy controls. A numerical increase in plasma exosomes level was described in patients with exacerbations compared to stable COPD patients. Moreover, the level of circulating exosomes correlated with plasma levels of CRP (p < 0.001), sTNFR1 (p < 0.01), and IL-6 (p < 0.01).

Higher levels of plasma CRP and sTNFR1 were observed in patients with exacerbations compared to stable COPD patients (p < 0.01 and p < 0.05, respectively) and healthy controls (p < 0.001 for both correlation). In stable COPD patients, plasma CRP was higher than in healthy controls (p < 0.001). Also IL-6 plasma level were higher in patients with exacerbations and stable COPD patients, compared to healthy controls (p < 0.05 and p < 0.01, respectively).

Overall, the study by Tan et al. (Tan et al., 2017) suggested that circulating exosomes were elevated in both stable COPD and in patients with COPD exacerbations, and a correlation with systemic inflammatory biomarkers was demonstrated.

In 2022, Meng et al. (Meng et al., 2022) described, in an in vitro and in vivo PM_2.5 exposure models, the effect of PM_2.5 on exosomal hsa_circ_0005045. PM_2.5, an ambient fine particulate matter, has been associated with higher prevalence of COPD among non-smoking subjects (Wang et al., 2018). The study showed that PM_2.5 induced and upregulated the exosomal circRNA hsa_circ_0005045. This molecule, through the binding to the exosomal protein peroxiredoxin2 (PRDX2), induced elastase (ELANE) and tumor necrosis factor (TNF)-α release by inflammatory cells, thus exacerbating airways inflammatory response.

A cohort of 83 males and 15 females, diagnosed with stable mild-to-moderate COPD, was enrolled. A pair of blood samples, pre-exposure and post-exposure, was collected from the same subjects at 10–14 days after an air pollution episode (daily PM_2.5 > 75 μg m^{− 3}). Eight matched blood samples from this cohort were used for circRNA microarray analysis and exosomes were isolated from blood before and after PM_2.5 exposure.

Healthy donors matched with sex, age, and smoking status were enrolled. Peripheral blood samples were collected during the stable phase in COPD patients and from healthy donors. Non-smoking was defined as smoking cessation for at least 1 year, and smokers were considered as subjects currently smoking or as having smoked at least 100 cigarettes in one’s lifetime.

Concerning in vivo murine model, male C57BL/6 mice treated group received PM_2.5 for 2 weeks leading to COPD-like lesions after exposure. Mice control group received high-efficiency particulate air-filtered room air (FRA) at the same flow rate.

CircRNAs microarray analysis showed that the levels of hsa_circ_0005045 in the plasma of COPD patients were significantly elevated compared to healthy controls. From circRNAs microarray analysis, performed on plasma samples of non-smoking COPD patients who experienced exacerbation after PM_2.5 ambient exposure, 111 upregulated circRNAs and 69 downregulated circRNAs were detected. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) was performed to confirm the upregulation of 6 specific circRNAs, including hsa_circ_0005045. Four of these circRNAs were associated with the “enrichment of extracellular exosomes” category, suggesting a correlation between circRNAs and exosomes. Analysis of the expression levels of 4 candidate exosomal circRNAs in 98 matched samples revealed that only hsa_circ_0005045 showed a significant upregulation in exosomes following exposure to PM_2.5. hsa_circ_0005045 potentially interacted in a complex with PRDX2 and neutrophil ELANE within exosomes. Both, PRDX2 and ELANE exhibited elevated plasma concentrations following PM_2.5 exposure, and these changes correlated with the levels of hsa_circ_0005045. Furthermore, hsa_circ_0005045 expression increased in exosomes derived from both bronchial and alveolar epithelial cells after in vitro exposure to PM_2.5.

A second cohort of 327 retirees diagnosed with COPD and divided in ABCD group according to GOLD 2017 (Barnes, 2017), was recruited to predict the risk of COPD acute exacerbations, by using a machine learning model. This model showed that COPD patients sensitive to PM_2.5 exposure were non-smoking, group C, and more likely to express higher levels of exosomal hsa_circ_0005045.

The function of hsa_circ_0005045 was further investigated in vivo, in a murine model with COPD-like lesions induced by PM_2.5 inhalation. The levels of a circRNA homologous to hsa_circ_0005045 were consistently and significantly elevated after 7, 14, and 28 days of PM_2.5 inhalation in COPD-like murine BALF and plasma-derived exosomes. Moreover, these exosomes, isolated from PM_2.5-inhaled mice, induced hallmarks of COPD and increased plasma levels of hsa_circ_0005045 homologous in healthy murine lungs. After PM_2.5 exposure, a significantly increase of PRDX2, inflammatory cells, and TNF-α was observed in lung tissues of both exposed and control mice, suggesting a potential contribution of the hsa_circ_0005045 homologous enriched exosomes to induce hallmarks of COPD after PM_2.5 exposure.

Recently, Jiang et al. (Jiang et al., 2023) conducted a preclinical study, in a mouse model of COPD, with the aim to investigate the role of serum exosome-derived miR-7 in the pathogenesis of COPD. miR-7 is a MicroRNAs considered a COPD biomarker, significantly upregulated in the serum of COPD patients (Akbas et al., 2012). The study showed that the level of miR-7 was significantly (p < 0.0001) increased in exosomes derived from COPD mice and in lung tissue macrophages (p < 0.01). Furthermore, COPD-exosomes induced an inflammatory response in the lungs of control mice. Lung tissue macrophages were found to be increased and showed elevated inducible nitric oxide synthase (iNOS) and decreased Arg1 levels, indicating a shift towards M1 macrophage polarization (iNOS, p < 0.0001; Arg1, p < 0.001). Moreover, pro-inflammatory cytokine levels secreted by macrophages showed significant increases (IL-6, p < 0.0001; TNF-α, p < 0.0001). The study also tested the effect of miR-7 inhibitor that blocked M1 polarization of macrophages and reduced IL-6 and TNF-α secretion, confirming miR-7 involvement in macrophage activation and differentiation.

Overall, the study showed that elevated miR-7 levels in the serum of COPD mice could play a role in COPD exacerbation by promoting M1 polarization in lung macrophages.

A prospective cohort study, available only in ClinicalTrials.gov (NCT04183530), aimed to perform a comprehensive characterization of COPD, through multidimensional data, including exosomes evaluation (ClinicalTrials.gov, 2023b). However, no results are still available.

In a study by Torregrosa Paredes et al. (Torregrosa Paredes et al., 2012), phenotypical and functional characteristics of BALF exosomes were investigated in asthmatic (n = 12) and healthy (n = 13) subjects. BALF exosomes were collected from mild allergic asthmatic patients, with birch pollen specific IgE (>2 kU/l), before and 24 h after birch allergen provocation. BALF exosomes from asthmatics showed an altered phenotypic profile, compared to healthy controls, even before allergen challenge. More specifically, higher levels of the surface molecules, such as tetraspanins CD81 and CD63, and HLA-DR were observed in BALF exosomes from asthmatic patients, compared to healthy controls. The scavenger receptor CD36, known to have a function in bacterial recognition (Baranova et al., 2008), and potentially implicated in asthma exacerbations in response to bacterial infections, was higher expressed by BALF exosomes from asthmatics, compared to healthy controls. No phenotypic changes were induced by allergen provocation, except for MUC1 that decreased after challenge. Both BALF exosomes from healthy controls and asthmatics expressed leukotriene A₄ hydrolase (LTA₄H), leukotriene C₄ synthase (LTC₄S), FLAP, and 15-LO-1 that are responsible of LTA₄ conversion to LTB₄ and LTC₄. Furthermore, after 48 h incubation, BALF exosomes of asthmatics induced significantly higher production of LTs and IL-8 in bronchial epithelial cells (BEC), compared to healthy controls, with no difference after allergen challenge. Only a modest increase in IL-6 production in BEC was observed, but with no significant differences between asthmatics and healthy controls.

The evidence of a pro-inflammatory altered exosome profile led to hypothesize a potential role of BALF exosomes in asthmatic inflammation.

In a preclinical mice model of asthma, Yu et al. (Yu et al., 2021) observed that ovalbumin (OVA) challenge of airways epithelial cells (AECs) induced the release of a major number of exosomes, compared to PBS-challenged AECs. OVA-challenged AEC-derived exosomes (OAEs) presented a different protein composition compared to PBS-treated AEC-derived exosomes (PAEs). More specifically, PLXNB2, a CD100 ligand, was the most expressed protein in OAEs and was found to be increased in BALF exosomes from asthmatic mice compared to controls. The proteolytic cleavage of macrophages CD100, mediated by OAEs MMP14, promoted pro-inflammatory responses in the airways, suggesting a potential mechanism of OAE-mediated asthma exacerbations. Interaction between CD100 and PLXNB2 allowed to internalize OAEs, representing a trigger to the transcription of pro-inflammatory chemokines and cytokines. Thus, OAEs were found to be able to increase airways hyper-responsiveness (AHR) and to induce the infiltration or activation of macrophages, neutrophils, and eosinophils in the airways. These observations highlighted the complex interaction between AECs and innate immune cells, powered by exosomes, in the pathogenesis of asthma.

Conversely, a preclinical study conducted by Song et al. (Song et al., 2020), evidenced the contribution of mesenchymal stem cell (MSC)-derived exosomes in the inhibition of chronic allergic inflammation, airways remodeling, and epithelial-mesenchymal transition (EMT) of airways epithelium in an asthma rat model. MSCs and MSC-derived exosomes significantly reduced inflammatory cells, such as lymphocytes, eosinophils, and neutrophils in OVA-sensitized and challenged rats’ airways. Moreover, treatment with MSCs and MSC-derived exosomes significantly reduced the proliferation of goblet cells and collagen deposition. All these effects were supposed to be mediated by exosomes highly expressed miRNAs affecting the expression of key proteins of the Wnt/β-catenin signaling pathway, implicated in EMT and airways remodeling. Indeed, treatment of rat model with BML-284, a small molecule agonist of the Wnt/β-catenin signaling pathway, reversed the downregulation of this pathway in airways epithelium, induced by MSCs and MSC-derived exosomes, exacerbating airways remodeling.

Recently, Wang et al. (Wang et al., 2022) conducted a longitudinal bi-phasic case-control study to characterize the profile of circulating exosomal microRNAs (ExoMiRNAs) in mild asthmatic patients and healthy controls, after in vivo rhinovirus (RV) challenge. RV is a common-cold-causing respiratory virus, notably cause of asthma exacerbations (Gern and Busse, 1999). Mild allergic asthmatic patients (n = 12) and matched non atopic healthy controls (n = 12) were recruited. Serum samples were collected before and after RV challenge. Inflammatory markers were also evaluated, including cytokines, chemokines, eosinophils, and neutrophils in nasal lavage. Furthermore, fractional exhaled nitric oxide (FeNO) and pulmonary functions parameters were measured. At baseline, no difference in miRNA expression was observed between asthmatics and healthy controls. After the RV challenge, a total of 26 ExoMiRNAs were differentially expressed (DE) between asthmatics and healthy controls. Expression of these 26 DE ExoMiRNAs not only was different before and after RV challenge in asthmatics, but it was differentiated also from miRNA expression in healthy controls, after RV challenge. Among these miRNAs, two clusters were identified. The Upregulated Cluster miRNAs, that significantly correlated with Th1 and regulatory cytokine, and Downregulated Cluster miRNAs, that significantly correlated with pulmonary function measurements, inflammatory biomarkers, and Th2 and Th17 cytokine groups. Overall, the study described the regulatory roles of ExoMiRNAs in cytokine-mediated immune response, in RV-exacerbated asthma.

A randomized study, available only in ClinicalTrials.gov (NCT04542902), was designed to perform a characterization of eosinophils derived exosomes in asthmatic and severe asthmatic patients, with the aim to investigate the role of different eosinophils subtypes in asthma pathogenesis and to further delineate asthma phenotypes (ClinicalTrials.gov, 2023a). To date, no results are still available.