Acute respiratory distress syndrome (ARDS) is a heterogeneous entity associating clinical, radiological, and pathological features leading to a severe decrease in lung diffusing capacity and compliance (Ashbaugh et al., 1967). ARDS is characterized by diffuse alveolar damage (DAD), associated with an increase in alveolar and capillary permeability resulting in an interstitial and alveolar edema that finally requires mechanical ventilation (Katzenstein et al., 1976; Mendez and Hubmayr, 2005; Cardinal-Fernández et al., 2017). The diagnosis of ARDS is based on the Berlin criteria (2012), stating that clinical presentation arises within seven days after exposure to a “common risk factor,” with pneumonia being the major cause (ARDS Definition Task Force et al., 2012). In up to 8% of ARDS cases, the leading cause of pathology is undetermined despite a systematic diagnosis approach, resulting in increased mortality rate. Thus, a methodical comprehensive work-up is necessary for ARDS of unknown origin.

The diagnosis is based on the association of DAD with deep hypoxemia. The prevalence is variable according to epidemiological studies but could reach up to 10.4% in patients admitted to the intensive care unit for a mortality of around 50% in the subgroup of patients suffering from severe ARDS (Bellani et al., 2016). In addition, survivors are at high risk of developing cognitive decline, depression, post-traumatic stress, and the classic side effects of long-term care, such as polyneuropathy, sarcopenia, or stroke disorder, sleep. In this context and in spite of significant progress in the field of mechanical ventilation and extra-corporeal respiratory assistance, it remains essential to identify early patients with ARDS in order to offer them the most appropriate therapy as soon as possible.

The lesion initially found in ARDS is the appearance of lesional edema resulting from an increased permeability to liquid and protein across the lung endothelium. Increased alveolar–capillary permeability results in accumulation of protein-rich fluid inside the alveoli, thereby producing diffuse alveolar damage (Bachofen and Weibel, 1982; Matthay et al., 2012). Then, the activation of resident alveolar macrophages will induce production of large amount of pro-inflammatory cytokines (interleukin (IL)-6, IL-8, tumor necrosis factor-a) at the origin of the recruitment of polynuclear neutrophils to the alveolar compartment and amplification of the inflammation response (Potey et al., 2019). Classically after 72 h, the repair processes are initiated, under the assumption of control of the etiopathogenic phenomenon at the origin of the lesion process. During this phase, there is intra-alveolar and capillary migration of (myo)fibroblasts and other progenitor cells with the aim of restoring the alveolar–capillary barrier ad integrum (Zemans et al., 2011; Dial et al., 2017). Once epithelial integrity is restored, the edema is resolved. The last phase is the recovery phase associating repair and scarring processes including fibroblastic activation. This fibrotic phase is particularly associated with a high morbidity–mortality rate due to its irreversibility and the association with an increased ventilation time. In this context, there is a high need for specific biomarkers helping clinicians to predict the evolution of patients (Thompson et al., 2017).

MicroRNAs (miRNAs) are small non-coding RNAs (19–22 nucleotides) which play a crucial role in the regulation of gene expression by repressing the translation of target genes or degrading their target messenger RNAs (mRNAs) (He and Hannon, 2004; Cheng et al., 2014; Njock et al., 2015; Guiot et al., 2020). miRNAs are secreted into body fluids (e.g., bronchoalveolar lavage fluid, saliva, sputum, and plasma) within extracellular vesicles, including exosomes (30–150 nm). The encapsulation of exosomal miRNAs into a bilayer lipid membrane protects them from degrading enzymes (e.g., ribonucleases) and confers them a remarkable stability in the bloodstream (Valadi et al., 2007; Li et al., 2015). The composition of exosomal miRNAs varies according to the state of their parental cells. In the pathological context, exosome content is robustly altered, including miRNAs, which represent a promising source of biomarkers (Guiot et al., 2019, 2020). Recently, Njock et al. (2019) identified a unique signature of three altered miRNAs (miR-142-3p, miR-33a-5p, and Let-7d-5p) from sputum of IPF patients. Several studies have reported an alteration of inflammatory-related exosomal miRNAs from airway biofluids (BALF and sputum supernatants) in the asthma context (Maes et al., 2016; Suzuki et al., 2016). In ARDS, only few studies have investigated the identification of altered circulating miRNAs, particularly exosomal miRNAs, and most of the works have focused on animal models, limiting knowledge on humans (Rahmel et al., 2018; Xu et al., 2020). Furthermore, there is no study on the characterization of exosomal miRNA content in ARDS. It is a systemic inflammatory disease, which suggests that blood exosomes generated by circulating cells (e.g., platelets and immune cells) as well as tissue cells (e.g., pulmonary, cardiovascular, renal, and hepatic) may participate in the pool of altered miRNA characteristics of ARDS.

In this study, we aimed to characterize small RNA content of plasma exosomes from ARDS patients in order to identify potential diagnostic biomarkers of the disease. For this, we profiled miRNA expression levels in plasma-derived exosomes from ARDS patients compared to healthy subjects (HSs) by small RNA sequencing (RNA-seq). This allowed us to identify nine differentially expressed (DE) miRNAs (miR-122-5p, Let-7d-3p, miR-24-3p, miR-130a-3p, miR-98-3p, miR-221-3p, miR-193a-5p, miR-1273a, and Let-7a-5p) in the ARDS context (in both screening and validation cohorts). The evaluation of their diagnostic potential showed that these DE miRNAs differentiate ARDS patients from HSs with a high level of accuracy. This study provides candidate biomarkers for the diagnosis of ARDS disease and opens new perspectives for the development of therapeutics.

ARDS is a common cause of respiratory failure in critically ill patients, induced by acute inflammatory response in the lung. As the diagnosis and treatment of ARDS is complex, there is a need to develop novel biomarkers to identify early patients in order to deliver more appropriate treatment and new potential therapeutic targets. Here, for the first time, we characterized miRNA content of plasma exosomes from ARDS patients by small RNA-seq. We identified and validated the alteration of nine miRNAs in plasma exosomes from ARDS patients compared to HSs. ROC curve analysis showed that seven of them could discriminate ARDS patients from HSs with a high level of accuracy (AUC>0.8): miR-130a-3, miR-221-3p, miR-24-3p, miR-98-3p, Let-7d-3p, miR-1273a, and miR-193a-5p (Figure 4). These findings highlight exosomal miRNA dysregulation in the plasma of ARDS patients and provide candidate biomarkers for the diagnosis of ARDS disease.

In ARDS, only a few studies have investigated the identification of altered circulating miRNAs. Rahmel et al. (2018) have shown that circulating miR-122 is elevated in ARDS patients compared to controls and associated with mortality and acute liver injury. In another study, Xu et al. (2020) have shown that serum miR-92a expression is higher in patients with sepsis-induced ARDS, when compared to patients with sepsis only. Recently, Zhu et al. (2017a) profiled miRNA expression in blood preparation from ARDS patients and critically ill at-risk controls and identified three altered miRNAs (miR-424, miR-92a, and miR-181a). To date, no study has characterized miRNA content from exosomes of ARDS patients. There is just one study where the levels of selected miRNAs from blood exosomes of ARDS patients were assessed. Indeed, Wu et al. (2019) investigated the expression of ten ARDS-relevant miRNAs (obtained by literature search) from the serum of patients with/without ARDS and identified a subset of four altered miRNAs (miR-146a, miR-27a, miR-126, and miR-155).

To our knowledge, our study is the first to profile miRNAs from plasma exosomes of ARDS patients by small RNA-seq in order to identify the ones that are the most altered. We identified several miRNAs that have already been described as potential biomarkers or involved in the development of ARDS (miR-122-5p (Rahmel et al., 2018), miR-221-3p (Zheng et al., 2018), miR-486-5p (Luo et al., 2020), miR-24-3p, and Let-7a-5p (Huang et al., 2014)), but we have also some miRNAs that have not yet been associated with the disease (miR-130a-3p, miR-193a-5p, or miR-1273a) (Figures 1, 3). The functional study of these miRNAs could open new avenues for the development of therapeutics.

IPA revealed that most of ARDS-related miRNAs are associated with different processes implicated in the pathobiology of the disease, mostly inflammation of the body cavity (p = 7.09E-07) and organ (p = 7.26E-06) (Figure 2B). Interestingly, there are few overlapping miRNAs in ARDS and IPF. Indeed, two altered miRNAs have been associated with IPF: miR-142-5p and Let-7a-5p (Figure 2B). Yang et al. (2015) have shown that Let-7a-5p was significantly decreased in the serum of IPF patients compared to HSs. Another study revealed that miR-130a-3p and miR-142-5p, which were dysregulated in macrophages from tissue samples of IPF patients, were able to sustain the profibrogenic effect of macrophages and therefore might be involved in the development of pulmonary fibrosis (Su et al., 2015).

Recent studies confirmed involvement of these miRNAs in the induction of ARDS-related processes. miR-122, mainly from hepatic origin, plays a role in the regulation of the hepatocyte by controlling cellular proliferation, differentiation, and apoptosis (Jopling, 2012). Furthermore, it has been shown that miR-122 is a specific serum biomarker for hepatocyte damage (Verma et al., 2016). Moreover, the association of APACHE II score and elevated serum miR-122 expression level is associated with disease severity and prognosis of ARDS patients (Hao et al., 2019). The functional study of miR-122-5p in the LPS-induced acute lung injury (ALI) mouse model revealed that a high level of miR-122-5p acted as a pathologic factor that exaggerated lung injury, inflammation, and oxidative stress and promoted ALI progression (Lu et al., 2020). Thus, miR-122-5p inhibition may be a potential treatment strategy for ALI caused by Gram-negative bacterial infection. To conclude, this upregulated biomarker is therefore above all an element of severity in our cohort, reflecting states of multiorgan failure.

miRNAs have been reported to be involved in the inflammatory response in many ways. With regard to miR-221-3p, its upregulation has been shown to play a role in the inflammatory response and the development of ALI in an LPS-induced mice model (Wang et al., 2019). Furthermore, increasing evidence suggests that extracellular vesicle–enriched miR-221-3p is the key factor in the pathogenesis of ARDS. Indeed, these vesicles actively delivered miR-221-3p into alveolar macrophages, subsequently promoting inflammasome activation, neutrophil recruitment, and M1-macrophage polarization (Lee et al., 2016, 2017; Zhu et al., 2017b). Another study reported that miR-221-3p induces the shift of M2-macrophages to a pro-inflammatory phenotype (Quero et al., 2020). The inhibition of miR-221-3p could therefore have a probable protective effect on ARDS progression.

miR-24-3p has also been implicated in ARDS. Unlike the work carried out on ARDS models in rats (Huang et al., 2014), we note that miR-24-3p is upregulated in our cohort. Moreover, it has been shown that overexpression of miR-24-3p significantly reduced LPS-induced lung damage and decreased the release of pro-inflammatory cytokines in the rat model (Lin and Yang, 2019). This complexity between pro-inflammatory and anti-inflammatory balance must still be understood in vivo.

It has been clearly demonstrated that the upregulation of miR-130a-3p plays a role in the differentiation of cardiac myofibroblasts by targeting the PPAR gamma pathway (Li et al., 2017). We can therefore imagine that this pathway is also involved in balance fibrosis/anti-fibrosis occurring in ARDS disease.

The downregulation of Let-7a-5p has been observed in a previous study in the rat model of ARDS (Huang et al., 2014). Furthermore, Let-7a-5p has been reported to suppress cell growth in multiple cancer types (Zhao et al., 2018); however, the pathophysiological mechanism is not yet known. One study suggests that Let-7d-3p exerts protective effects on cardiac cells subjected to hypoxia stress by inhibiting apoptosis (Wong et al., 2019). Future work has to be carried out in order to specify the role of Let-7a-5p and Let-7d-3p in ARDS development.

miR-193a-5p was found to be upregulated in a mouse model of ALI induced by inhalation of staphylococcal enterotoxin B. Pathway analysis revealed that miR-193a-5p is involved in ALI progression by targeting several molecules of transforming growth factor-beta (TGF-β) signaling (TGF-β2, TGFβ-R3) and apoptotic signaling pathways (Alghetaa et al., 2018).

To decipher molecular mechanisms that could contribute to the disease, we investigated signaling pathways that may be impacted by ARDS-miRNAs. The main pathways targeted are STAT3, PI3K/AKT, MAPK, and NF-kB pathways (p < 0.05) (Figure 2). Interestingly, several studies have shown that these signaling pathways are involved in the development of ARDS. Indeed, the impact of NF-kB on ARDS progression is well studied (Meduri et al., 2005; Hariharan et al., 2020), as well as PI3K/AKT (Y et al., 2020) and MAPK (Newton and Holden, 2006). Recently, Zhang et al. (2019) found that STAT3 may be the key regulatory gene in the underlying dysfunction of sepsis-induced ARDS. The mRNA targets associated with ARDS-miRNAs are potential candidates for development of new therapeutic strategies.

In conclusion, we identified potential biomarkers for the diagnosis of ARDS from plasma-derived exosomes. From the nine altered miRNAs identified, seven present a good capacity to discriminate ARDS patients from HSs. These miRNAs are involved in different biological processes associated with the development of the disease such as inflammation or fibrosis. The biomarker status of these exosomal miRNAs needs to be validated in large independent cohorts. In addition, their potential value on early detection of ARDS needs to be evaluated in future studies. This study provides the basis to improve ARDS diagnosis and open avenues to develop new therapeutic strategies.