**3. Asthma Exacerbation and miRNAs**

MiRNA molecules perform various functions in the body. In addition to the diagnostic role described above, they can be used to predict exacerbation of the disease with greater accuracy. The risk of exacerbation of the disease increases with the lack of use of inhaled corticosteroids, lack of adequate disease control, spirometric indices indicating deepening of obstruction, signs of eosinophilic inflammation, or exposure to allergens or irritants. In a group of children diagnosed with asthma and using anti-inflammatory treatment (inhaled steroids) for a period of 12 months, 12 miRNA molecules associated with exacerbations during observation were isolated. In addition, each doubling of the concentration of these molecules was associated with an increase in the risk of exacerbations by 25–67%, respectively. MiRNA-146b-5p, miRNA-206, and miRNA-720 were identified, which, in combination with clinical symptoms, enabled a better prediction of exacerbation of the disease in patients with asthma using inhaled corticosteroids, compared to a model based only on a single component. Involvement of these miRNAs in pathways involved in the pathogenesis of asthma was discovered, e.g., NF-kβ and GSK3 (glycogen synthase kinase-3)/AKT, which are responsible for remodeling the airways and deepening the inflammatory process [2].

MiRNAs are biomolecules that also play a key role in other obstructive respiratory diseases, including chronic obstructive pulmonary disease (COPD). Researchers compared circulating serum miRNAs associated with exacerbation of the disease in the pediatric population diagnosed with asthma and adults diagnosed with COPD. A total of 20 miRNA molecules were associated with worsening of symptoms in children and 5 of them (451b; 7-5p; 532-3p; 296-5p, and 766-3p) in the adult population with severe chronic obstructive pulmonary disease. Participation of these molecules in the signaling pathways MAPK (mitogen activated protein kinases) and PI3K-Akt, which are responsible for the increased response of both eosinophils and neutrophils, the production of immunoglobulin IgE, and the activation of tumor necrosis factor alpha (TNF-a), was proven. This leads to excessive production of interleukins, cytokines, and intensification of inflammation [24]. In addition, the MAPK pathway is activated by tobacco smoke, indicating its important role in the pathogenesis of COPD and neutrophilic asthma. Thanks to the analysis of the concentrations of appropriate miRNAs, it is also possible to identify the pathomechanism that may lead to exacerbation of symptoms among children with asthma who are exposed to tobacco smoke in the home environment. This is undoubtedly a factor that stimulates an increased immune response and affects the disease symptom severity. However, anti-inflammatory molecules have been identified that are also related to the concentration of the relevant miRNAs. One of them is annexin (ANXA1), also known as lipocortin I. It is a protein with a significant content in the secretions of the respiratory tract. It was shown that an increase in ANXA1 concentration was associated with the decreased concentration of miRNA-196-a2. It is also interesting that in subjects with a moderate disease severity, the level of ANXA 1 was higher than in those with severe asthma. In addition, miRNA-196-2a is responsible for the production of key interleukins involved in the pathogenesis of asthma, IL-5 and IL-13, whose role is described above [25–27]. Many factors can exacerbate the course of asthma, such as tobacco smoke, infections (mainly viral), allergens, irritants (e.g., aerosols or household cleaners, paint fumes, other occupational exposures), physical exertion, air pollution, medications, and foods. Respiratory infections caused by viruses, including RSV, influenza, and rhinoviruses, are of particular importance among the group of the youngest patients. When these pathogens infect human bronchial epithelial cells (HBECs), it was demonstrated that the NF-κB pathway and interferon signaling are activated to stimulate cellular responses, reduce viral replication, and avoid tissue damage. It was suspected that the HBEC cells of asthmatics impair the above-described processes and, consequently, exacerbate symptoms. Understanding miRNAs targeting the NF-κB and interferon pathway will uncover modulators of cellular responses and prevent the development of adverse events [28]. MiRNA-146a and miRNA-146b (miRNA-146a/b) are anti-inflammatory molecules more heavily produced in response to rhinovirus (RV) infection, targeting the NF-κB pathway specifically. In experimental animal models of allergic asthma, deprivation of HBEC cells of these miRNAs led to an increased inflammatory process involving Th1, Th17 lymphocytes with a reduced participation of Th2 lymphocytes. These molecules are therefore responsible for alleviating RV-induced allergic airway inflammation and represent a potential future therapeutic target [29]. Influenza virus is another important infectious agent responsible for exacerbating asthma symptoms. After infection of bronchial epithelial cells with H1N1 influenza virus in the laboratory, miRNA-22 growth was demonstrated only in a sample obtained from healthy individuals. The molecule blocks the CD147 receptor which is a transmembrane glycoprotein involved in the invasion of viral and bacterial infections. This molecule also participates in the remodeling of the respiratory tract through the increased synthesis of matrix metalloproteinases (MMPs). Therefore, asthma patients with low levels of miRNA-22 due to the inability to block CD147 lose one of the important defense barriers against viral infection, becoming more susceptible to infection. In the era of the COVID-19 pandemic, it was shown that the CD147 receptor is also responsible for the penetration of the SARS-CoV-2 virus into the cell. This proves the role of miRNA-22 in the defense process against a wider spectrum of viral infections than just influenza [30].

Unfortunately, not only infectious factors play a leading role in the pathogenesis of asthma in children, but also increasing air pollution and high concentrations of harmful substances in the environment. After measuring the content of PM 2.5 particles in the composition of the air at home, a significant correlation was shown between their concentration and the incidence of asthma. Moreover, greater exposure resulted in an increase in serum concentrations of miRNA-155, which is responsible for the enhancement of the type 2 immune response [31]. The relationship of this biomolecule was also confirmed with exposure to tobacco smoke, which is a special type of air pollution due to its local occurrence and relative ease of elimination from inhaled air compared to other factors. MiRNA-155-5p, miRNA-21-3p, and miRNA-18a-5 are a set of molecules that are overexpressed in the blood of mice exposed to cigarette smoke in utero, in which asthma was later induced with albumin. Additionally, their concentrations were positively correlated with proasthmatic Th2 cytokine levels in bronchoalveolar lavage fluid (BALF) samples [32]. Another aspect that was emphasized was the seasonal variability of miRNAs and its impact on the severity of the allergic process and asthma symptoms in the pediatric population, especially in the spring. It is at this time of the year patients are at risk of exacerbating asthmatic symptoms, especially those with hay fever. Of the 26 miRNAs studied, which were associated with a specific season and with the allergen causing the symptoms of this period, two miRNAs-328-3p and let-7d-3p were isolated. A decrease in the concentration of let-7d-3p was observed in spring and among those allergic to mulberry (spring blooming), while it increased after the allergen immunotherapy process (performed in patients allergic to wasp venom). The protective role of this molecule was proven, which is most likely achieved by reducing the concentration of IL-13. Its protective effect in relation to asthmatic patients is also supported by its abundant presence in the lung tissue. The role of miRNA-328-3p is more complex. It was observed that its concentration decreases in autumn. On the other hand, an increase was noted in patients with a concomitant allergy to aspergillus, which resulted in an exacerbation of symptoms. MiRNA-328-3p was shown to be involved in wound healing of the bronchial epithelium, but also facilitates the spread of bacterial infection in the lungs [33].

MiRNAs may participate in the process of airway remodeling, leading to airway obstruction and affecting airway function parameters. MiRNAs involved in a number of pathophysiological processes affect smooth muscle cells, the epithelium, and goblet cells and intensify the inflammatory process, causing narrowing of the bronchial lumen [34].

The relationship between 22 miRNAs and lung function parameters in the pediatric population was demonstrated. An example may be the following molecules: miRNA-186-5p, which participate in the release of acetylcholine and modulation of airway tone through the cholinergic pathway [35,36]; miRNA-203, which is associated with an increase in IgE concentration and intensifies the inflammatory process in the airways, leading to their obstruction [35,37]; and miRNA-26, which is released by bronchial smooth muscles after their physical stretching, causing cell hypertrophy. Over the course of many years of observation, it has also been proven that miRNA-145-5p is associated with an early decrease in FEV1 in children with asthma, leading to the development of COPD. It is also responsible for the increased proliferation of airway smooth muscle cells [38].

The results of the studies on miRNAs in pediatric asthma are summarized in Table 1.

**Table 1.** The relevance of miRNA in asthma based on studies conducted in the pediatric population.



**Table 1.** *Cont.*

receptor subfamily three group C member 1 gene; PTEN—phosphatase and tensin homolog gene; GSK3—glycogen synthase kinase-3; AKT—protein kinase *B*; NF-kB—nuclear factor kappa-light-chain-enhancer of activated B cells; MAPK—mitogen-activated protein kinases, PI3K—the phosphatidylinositol 3-kinase; Ige—immunoglobulin E; GATA3—GATA binding protein three; Th1—Type 1 T helper; Th2—Type 2 T helper.
