**1. Introduction**

Asthma is characterized by chronic airway inflammation and airway hyperresponsiveness, it is a heterogeneous disease commonly seen in childhood as a chronic airway disorder [1]. The latest prevalence of childhood asthma in the United States is 5.8% [2], while there is a lack of unified and comprehensive epidemiological survey in China, the prevalence of childhood asthma in China in recent years is about 4.90% through meta-analysis [3], which was higher than the prevalence of the Third National Childhood Asthma Epidemiological Survey of Chinese major cities in 2010 [4]. The research documents the association of immunity with the development of asthma, which is currently believed to be an airway inflammatory disease dominated by a Th2 type of immune response. Found throughout the body in cells, tissues, and body fluids, MicroRNAs are noncoding endogenous RNAs of 19 to 25 nucleotides in number, which base-pair with target gene mRNAs to regulate posttranscriptional expression of the target gene by silencing or blocking the target gene. MicroRNAs are nonspecific and can simultaneously regulate several target genes to fulfill biological roles [5–7]. Their involvement in asthma development has been proven, among others, through

the promotion of T cell differentiation toward Th2, the increase of Th2 cytokines, and the decrease of Th1 cytokine secretion [8–11].

Recent studies have highlighted the importance of type 2 innate lymphocytes (ILC2s) in the development of asthma [12, 13]. ILC2s are primarily generated from common lymphoid progenitors (CLPs) in bone marrow, where their maturation proceeds and contributes to intrinsic immunity and tissue repair. It is generally assumed that Th2 cells are the primary source of type 2 cytokines. In contrast, more studies have surfaced that ILC2s can also be a significant early contributor to type 2 cytokines and that such cells can be critical in both the initiation and effector phases of type 2 immunity. ILC2s trigger both the innate response to allergic inflammatory responses and the immune response to adaptive Th2 [14–16]. Experimental stimulation of ILC2s-deficient mice with allergens revealed that the mice did not produce a solid allergic inflammatory response in the lungs and that Th2 cell differentiation was impaired; however, the impaired Th2 cell differentiation was rescued by ILC2 transplantation [15]. Therefore, ILC2s are essential for Th2 cell-mediated allergic lung inflammation and can induce the differentiation of CD4+ T cells into Th2 cells. Furthermore, ILC2s and Th2 cells interact reciprocally during the type 2 immune response, with either the interaction of co-stimulatory molecules or in a direct cell–cell contact-dependent manner through soluble mediators, such as cytokines [17, 18]. In response to the Th2 polarizing cytokines IL-25 and IL-33, ILC2s rely on transcription factors, such as RORα and GATA3, to differentiate and mature [19, 20]. Similarly, n response to IL-4, Th2 cells also depend on transcription factors, such as GATA3, for differentiation and maturation. After the activation of both ILC2s and TH2, a significant amount of Th2 cytokines, IL-4, IL-5, and IL-13 would be produced of which IL-25 is constitutively expressed by clustered cells in the intestine and remains essential for ILC2s to remain stable, IL-25 induces IL-13 production by ILC2s, and IL-13 production by ILC2s and/or Th2 cells may conversely promote differentiation and expansion of clustered cells, creating positive feedback [15, 21, 22]. MiRNAs are increasingly known to regulate the activation of ILC2s and Th2 and Th2-related inflammatory factor production, leading to their involvement in the pathogenesis of asthma. Below is an overview of how miRNAs are involved in asthma development by mediating Th2 and ILC2s.

#### **1.1 Relation between MiRNAs and Th2 differentiation and homeostasis**

Th1 and Th2 are in relative homeostasis under normal physiological conditions, whereas an imbalance in the Th1/Th2 ratio and excessive secretion of Th2 cytokines contribute to the pathogenesis of allergic asthma. Most studies have documented that miRNAs are abnormally expressed and are strongly associated with disorders of the Th1/Th2 immune response in asthma [9, 10, 23, 24]. Up or down-regulation of miR-NAs can lead to increased secretion of Th2 cytokines or decreased secretion of Th1 cytokines. Up-regulated miRNAs in asthma include miR-21, miR-126, miR-221-3p [25], and miR-3162-3p [26]. Up-regulated miRNAs, including miR-451, miR-1165, miR-29b [27], and miR-135a [28], increased Th2 cytokine secretion include miR-146. As miRNAs can affect T cell differentiation, activation, and eosinophilic mast cell activation in allergic diseases, they could be used as a potential means to reduce Th2 inflammation from the altered genetic level and as noninvasive biomarkers for early prediction of asthma development. Of the many publications on how miRNAs regulate Th2 cells, a few of the most extensive studies are presented below.

### **1.2 MiR-155**

MiR-155 is an indispensable miRNA for functioning dendritic cells, T cells, B cells, and other immune cells. In the past, much attention has been focused on miR-155's involvement in pro-inflammation and Th1 immunity, and it is labeled as a "Th2 inhibitor" [29]. When compared to WT mice, for example, miR-155(−/−) mice showed significantly lower numbers of inflammatory cells in alveolar lavage fluid, which compromised the initiation of Th2 responses and reduced airway inflammation [8]. With further studies, however, miR-155 shows an integral contribution to allergic asthma. Therefore, in patients with asthma, there is decreased expression of miR-155 in peripheral blood CD4+ T cells, notably in severe asthma [30], meaning it is strongly associated with asthma severity, which may be achieved by directly targeting the IL-13 pathway suggesting that a strong correlation between miR-155 and asthma severity exists, which is probably attained by directly targeting the IL-13 pathway [31]. Compared to WT mice, miR-155-deficient mice have significantly reduced eosinophil content in alveolar lavage fluid after allergen stimulation [32], demonstrating that Mir-155 may gather more eosinophils during airway inflammation. In a similar vein, Kim constructed mouse models of miR-155 deficiency in T cells and identified lower levels of Th2 cytokines in alveolar lavage fluid and lower mucus secretion in the experimental group compared to the control group [33]. These findings recommend that miR-155 from T cells is essential for Th2 allergic airway inflammation as a crucial serological biomarker for asthma diagnosis and severity.

### **1.3 MiR-21**

Statistical studies of clinical data have found significantly higher levels of miR-21-3p and miR-487b-3p expression among the sera of children in the acute phase of food allergic reactions [34], presumably due to the targeted binding of lL-12 and miR-21-3p, which by down-regulating the former, could promote th2 inflammatory responses. Similarly, studies in an asthma rat model experiment revealed that miR-21-5p expression was significantly up-regulated, mainly in alveolar macrophages. MiR-21-5p produced by macrophages was confirmed by *in vitro* experiments to be targeted by the TGF-β1/Smad-signaling circuit to Smad7 after translocation to the airway epithelium for promoting EMT (epithelial-mesenchymal transition) [35], thereby enhancing airway remodeling leading to the development of asthma. Zhou demonstrated the involvement of mast cells in asthma development in an asthma model, where miR-21 was present in derived EV and could exacerbate the airway inflammatory response in mice via the DDAH1/Wnt/β-catenin axis [36]. Furthermore, a significant reduction in Th2 cytokine expression, an increase in IL-12 and IFN-γ secretion, and a reduction in cup cells in lung tissue were observed in miR-21-deficient asthmatic mice [37], which in turn attenuated airway inflammation as well as remodeling. From the above, miR-21 can be involved in the development and progression of asthma by promoting Th2 differentiation and airway remodeling through various inflammatory response signaling pathways. It can serve as a potential biomarker for allergic reactions.

### **1.4 let-7 family**

The let-7 family was found as a second microRNA, down-regulated in various tumors and also referred to as a tumor suppressor. However, there is some

controversy about the role of let-7miRNAs in asthma. IL-13, a critical Th2 cytokine, was also a direct target of let-7 microRNAs, some of which were suggested to play an anti-inflammatory role in the immune response. Manis agreed that let-7miRNAs possessed an anti-inflammatory action and concluded that let-7miRNAs reduced the inflammatory response by targeting the IL-13 3'UTR site, thereby lowering IL-13 levels through *ex vivo* experiments in asthma models [38]. Instead, Polikepahad has found a pro-inflammatory effect of let-7 in a mouse model of asthma [39], which may be associated with differences in experimental methods, making it even keener for further study by those to follow. In addition, it has been found that let-7 inhibitors can enhance the effect of airway smooth muscle on β2-receptor agonists by reducing β2-receptors downregulateion [40], which is more effective in asthma control.
