Asthma is a heterogeneous disease of the airways characterized by airway inflammation caused mainly by eosinophils, lymphocytes, and mast cells (1). Symptoms of asthma consist of cough, difficulty breathing, and wheezing, and specific disease manifestations are related to the inflammatory status that causes remodeling and bronchial malfunctioning (2, 3). Worsening of lung function is often related with poor responses to asthma treatment, which may indicate a requirement of a higher dose of corticosteroids, novel biological drugs, or even combined therapies (4). This clinically complicated phenotype is known as severe asthma, which carries a high economic and social burden (5), and is usually driven by eosinophilic inflammation. Patients with severe asthma require higher doses of corticosteroids, and novel biomarkers are needed to discriminate patients who present this phenotype, as doing so will facilitate clinical practice (6).

The heterogeneity of the asthmatic immune responses is a key element which has been a target of research since the first airway samples were taken until the newest technology clustering and bioinformatic analysis, depicting asthma subgroups that are related to severity, age of onset, treatment, and cellularity (7). The introduction of phenotypes, and more importantly endotypes, beneath severe asthma is one of the most important advances into how we understand asthma pathophysiology, and has been useful in defining, depending on the type of inflammation, the type 2 high severe asthma endotype (ILC2 and Th2 driven), the type 2 low endotype (neutrophilic), and the mixed endotype (T1/T2 and T2/T3 asthma) (8). Some of the differences seen between the asthmatics and the diverse phenotypes and endotypes inside this heterogeneous disease may be explained by epigenetic regulation, which acts in the development and mechanisms of asthma (9). One such mechanism involves the action of microRNAs (miRNAs), which are small non-coding RNA molecules that act as regulators of protein synthesis by gene repression, inhibiting protein translation (10), and as such are useful as biomarkers due to their high stability and relatedness to the disease (11). As part of the epigenetic posttranscriptional regulation landscape, microRNAs (including our miRNA of study: miR-144-3p) are able to modulate the pathophysiology of severe asthma, favoring the development of particular immune responses, and therefore being part of severe asthma endotypes (12).

Previous reports have described roles for miRNAs as asthma mediators and biomarkers (13, 14), and great potential has been shown for the latter (15) due to their possible use as biomarkers to facilitate asthma endotyping and phenotyping to assign treatments, as depicted in previous proof-of-concept studies (16, 17).

The objective of this study is to delve into the role of miR-144-3p, an miRNA previously related to asthma disease (18). In that first report, we observed that miR-144-3p was differentially expressed in blood eosinophils from asthmatics compared to those from controls. In this new report, we got more insight on miR-144-3p by studying its expression in the serum and lungs of asthmatics, stratifying the subjects depending on disease severity and treatment.

This study shows the relationship of miR-144-3p in asthma pathophysiology, and its derivative use as a disease biomarker. This claim is based on the finding of increased expression of miR-144-3p in lung biopsies and serum from asthmatics compared to controls.

Furthermore, the expression of miR-144-3p in bronchial samples from asthmatic subjects correlated directly with the expression of genes involved in asthma pathogenesis such as GATA3, STAT6, SOCS5, RHOA, NR3C1, and PTEN. GATA3, which was previously reported to be higher in airways from asthmatics (25); STAT6, a key molecule required for eotaxin production and pulmonary eosinophilia (26); SOCS5, which has been previously defined as an inductor of airway eosinophilia (27); RHOA, a kinase with the ability to control variable cell functions deregulated in asthma including proliferation, recruitment, and contraction (28); NR3C1, which is the coding gene for the corticosteroid receptor whose single-nucleotide polymorphisms (SNPs) are associated with reduced corticosteroid effect over TGF-β (29); and PTEN, a phosphatase that has been related to the successful treatment of airway inflammation and hyperresponsiveness (30).

The correlation observed between the expression of these genes and miR-144-3p may be indirect, resulting in the overexpression of these genes in asthmatic lung tissue. Having in mind that SOCS5 is a suppressor of cytokine signaling that is able to regulate negatively the activation of STAT6 induced by IL-4, these two genes may be involved in a negative feedback loop to control cytokine synthesis (31). Besides, miR-144-3p overexpression in bronchial smooth muscle cells only affected GATA3 mRNA, and previous reports have shown that the reduction of GATA3 by SB010 DNAzyme attenuated allergic asthma responses by type 2 helper T-cell (Th2) reduction (32), so in this sense, miR-144-3p may behave as an inhibitor of GATA3 in a similar manner and may even control signaling pathways involved in asthma pathophysiology as observed in silico. Nonetheless, this observation should be confirmed in further in vitro and in vivo severe asthma models, to get a complete insight into the role of miR-144-3p in severe asthma.

Several studies have attempted to use miRNAs for asthma severity determination in biofluids. MiR-28-3p, miR-16-2-3p, and miR-210-3p have recently been associated with severe asthma and were suggested as biomarkers, although the study was only preliminary (33). Other reports have elucidated profiles of miRNAs in sputum related to neutrophilic severe asthma, of which miR-223-3p was found to be a key node (17). In addition, our research group has previously described miRNA profiles that could be helpful in classifying asthmatics according to the severity of the disease, in discrimination of the eosinophilic phenotype, or for the prediction of the response to biological drugs (16, 18, 34, 35). This new report on miR-144-3p adds new knowledge of miRNAs in severe eosinophilic asthma, reporting another miRNA that can help in unravelling the complex epigenetic landscape of this disease.

The association of miR-144-3p with severe asthma was confirmed in serum, as miR-144-3p levels were higher in the severe asthmatic group compared to both the mild asthma group and healthy controls, showing that on the systemic level the expression of this miRNA is increased among patients with the most severe disease. These data are interesting as, in our previous study, we did not find any difference between healthy and asthmatics serum miR-144-3p expression, but indeed, when stratifying the asthmatics into severity phenotype, the difference can be seen (18). This highlights the importance of stratifying the asthmatic patients depending on different phenotypes or endotypes, as with this, some previously hidden characteristics can be found.

The relationship between miR-144-3p and severe asthma is further supported by the inverse correlation observed between miR-144-3p and FVC, which implies that the higher miR-144-3p is in serum, the worse the lung function, possibly due to higher airway obstruction (36). The relationship between miR-144-3p and asthma severity is also reflected in the direct correlation observed with daily inhaled corticosteroid doses. Additionally, a higher expression of miR-144-3p was found in asthmatic subjects who required a systemic corticosteroid treatment event, in subjects with more than five visits to the ICU, and in subjects treated with leukotriene receptor antagonist; all of these events are related to severe asthma, with high corticosteroid treatment and poor treatment response. Many of these subjects may benefit from a more personalized treatment for their severe asthma such as biological drugs, which can reduce asthma symptoms and corticosteroid dosage, thereby indicating that miR-144-3p expression analysis may help in identifying these patient candidates (37).

In accordance with our results, in another study, miR-144-3p was found increased in mouse lungs upon dexamethasone treatment (38), and indeed, we found that miR-144-3p expression in the lungs has a tendency for higher levels in asthmatics under corticosteroid treatment. Inside the group of asthmatics under corticosteroid treatment, there could be observed what seems to be two small clusters inside it, with four patients having the highest miR-144-3p expression. Although this group is very small, a possibility exists that there are asthmatics with very high miR-144-3p expression in their lungs that may comprise a different endotype inside the corticosteroid-treated asthmatics. Nevertheless, further experimentation and studies are needed in this matter, as from our results we cannot associate this small endotype to any other clinical characteristic of asthma. This raises the question of whether the increase of miR-144-3p derives from the severity of asthma or from the high corticosteroid dose used to treat asthmatic subjects (39). This question will likely be of interest for future studies, although in the meantime it is noteworthy that the expression of miR-144-3p in the serum of asthmatic patients can be used as a biomarker for asthma subphenotyping, as seen in the ROC curve analysis, where the expression of this miRNA in serum had an AUC of 0.63 for differentiating severe asthmatics form mild to moderate asthmatics (being a mildly poor biomarker), and an AUC of 0.74 when distinguishing asthmatics who are in treatment with corticosteroids and bronchodilators from those with on-demand bronchodilator therapy (being a good biomarker for asthma treatment). The importance of biomarkers for asthma diagnosis has been previously shown, as lung function tests are not always good, and therefore combinatorial biomarkers seem to be the most promising diagnostic tool in asthma (40).

Moreover, the possible increase of miR-144-3p in the lungs and serum due to corticosteroid treatment, as observed in mouse lung tissue (38), and based on the direct correlation observed between corticosteroid dose and miR-144-3p expression, might be an indicator of corticosteroid use and treatment adherence and thus might be helpful in determining which patients are truly taking the treatment. This information might help in isolating those asthmatics that are corticosteroid responders from those who might need higher doses or who may benefit from an alternative therapy (41).

By contrast, it is worth noticing that miR-144-3p expression in eosinophils from asthmatic patients is lower than healthy eosinophils (18). This can be explained by different reasons, as the study was done in peripheral eosinophils; furthermore, eosinophils normally comprise around >3% of the asthmatic airways cellularity (42), and lung tissue is a complex network with many cell types interacting, and thus, the expression of a miRNA can be divergent between the whole tissue and a cell type that is part of it. This has been previously shown in other tissues like the brain, where miRNAs have different expressions and effects depending on the cell type (43).

There are some limitations in our study: one is the absence of a clinical classification of lung tissue into severity, although a trend toward a higher miR-144-3p expression could be observed in asthmatic lungs of subjects treated with inhaled or systemic corticosteroids, which may indicate an association to severe asthma; the second is that the small number of lung samples can be blurring both the correlation results, as the expression of miR-144-3p in biopsies seems to correlate inversely with FCV, and the trend for increased miR-144-3p expression in corticosteroid-treated asthmatics, having non-statistically significant results, which therefore cannot be thoroughly confirmed. Nevertheless, tendency for a direct correlation was observed between miR-144-3p expression in lung and the counts of blood eosinophils and thus together with the FCV correlation may indicate that the asthmatic subjects with higher miR-144-3p expression in their lungs present the severe eosinophilic asthma phenotype.

In conclusion, these results highlight the importance of miRNAs in asthmatic disease, from their involvement in pathophysiology by means of modulation of key pathways to their use in diagnosis as biofluid markers. In this study, we found that miR-144-3p is upregulated in lung biopsies from asthmatic subjects compared to healthy individuals and that miR-144-3p expression is correlated with worst lung function and with type 2 inflammatory profiles in blood. Furthermore, miR-144-3p is increased in severe asthmatics treated with higher doses of corticosteroids, thus opening new possibilities for asthma diagnosis and treatment.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Comite de Etica de la Investigacion de la Fundacion Jimenez Diaz. The patients/participants provided their written informed consent to participate in this study.

VP conceived the manuscript and designed the study. JR-M performed the experiments and data analysis and had primary responsibility for writing the manuscript. MG-M, CL-S, and RG-L performed the experiments and obtained data. MV-M, SQ, and JS recruited patients and obtained samples and clinical data. All authors contributed to the article and approved the submitted version.

This work was supported by the Fondo de Investigación Sanitaria PI18/00044, FI16/00036, and FI19/00067; Ciber de Enfermedades Respiratorias (CIBERES); RTC-2017-6501-1 (Ministerio de Ciencia, Innovación y Universidades); and FEDER funds (Fondo Europeo de Desarrollo Regional).

S.Q. reports personal fees from AstraZeneca, Novartis, Sanofi, Boehringer Ingelheim, Teva, ALK, Mundipharma, GSK, Chiesi, and personal fees from Leti, outside the submitted work. J.S. reports having served as a consultant to Thermofisher, MEDA, Novartis, Sanofi, Leti, Faes Farma, Mundipharma, and GSK; having been paid lecture fees by Novartis, GSK, Stallergenes, Leti, and Faes Pharma; as well as having received grant support for research from Thermofisher, Sanofi, and ALK. V.d.P reports having served as a consultant to Astra Zeneca and GSK and having been paid lecture fees by both.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.