The Relationship between microRNAs, ILC2s and Th2 Cells

Abstract

Asthma is a common and chronic inflammatory disease. The pathogenic mechanism underlying asthma is complex. Many inflammatory cells have been recognized as involved in asthma, containing lymphocytes (T, B cells), ILC2s, eosinophils, and other types of immune and inflammatory cells. It is well-established that allergen-specific Th2 cells play a central role in developing allergic asthma. In addition, in recent years, increasing studies have found that ILC2s can contribute to the pathogenesis of asthma by promoting the immune response of Th2 and secreting Th2 cytokines. MicroRNAs (MiRNAs and MiRs) is involved in immune inflammation and can induce excessive secretion of Th2 cytokines. The regulation of miRNAs to their targeting genes plays an important role in the development of asthma. This chapter has discussed altered expression and functions of miRNAs in Th2 and ILC2s in asthma, in order to better understand the mechanics of pathogenesis of asthma, and provide potential miRNA diagnostic indicators and therapeutic targets.

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, 6, 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, 9, 10, 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, 15, 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 miRNAs 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.

1.5 MiR-126

The relative levels of miR-126 in the peripheral blood of children with asthma were found to be both elevated and significantly correlated with the severity of the disease and also negatively correlated with IFN-γ levels, which has great potential for diagnosis, especially in severe asthma, with an AUC of 0.909 [41], as one of the diagnostic biomarkers for asthma. The up-regulated levels of miR-126 were positively correlated with the severity of lung function [42], which may be associated with IL-13 [41]. But they have higher small airway reversibility [43]. Concerning animal experiments, Mattes found in an ovalbumin (OVA)-induced asthma model that miR-126 blockade led to enhanced expression of POU structural domain class 2 associated factor 1, one that activates the transcription factor PU.1, which alters TH2 cell function by negatively regulating GATA3 expression [44]. GATA3 may facilitate Th0 to Th2 differentiation. During the construction of the chronic airway inflammation model, the expression of multiple miRNAs, especially miR-126, increased in the airway wall early in the model establishment, and when administered with miR-126 antagonists, inhibited airway eosinophil recruitment. In contrast, later in the model construction, miR-126 expression decreased, and continued administration of miR-126 antagonists affected long-term chronic inflammation of the airway walls with little change [45]. These results indicate that miR-126 plays a significant role early in pathological evolution and that early intervention should be of little clinical significance if it reaches a terminal stage.

1.6 Other related miRNAs

Analysis of clinical data identified that children with asthma presented with reduced levels of miR-34a, miR-92b, and miR-210 secretion from airway epithelial extracellular vesicle (EV), while the decline may enable DCs to polarize Th2 cells, giving rise to the asthma phenotype [46]. The extraction of lymphocytes from asthmatic children yielded significantly lower levels of miR-451a than baseline, miRNA-451a with an FC of 4.6 and a p-value of 0.008 (asthma vs. control), and down-regulation of miR-451a was observed when CD4+ T cells were placed in Th2 differentiation medium and ETS1 upregulation, leading to the evidence that miRNA-451 inhibits Th2 differentiation by downregulating EST1 [47]. Furthermore, a significant reduction in type 2 allergic lung inflammation was observed in asthma model mice with upregulation of miR-1165-3p in mouse lung tissue, suggesting that miR-1165-3p could inhibit Th2 differentiation, which was achieved by directly targeting IL-13 and PPM1A [48]. In addition, miR-29b upregulation in an asthma model can modify the Th1/Th2 balance by inhibiting ICOS expression, thereby attenuating eosinophil recruitment in airway epithelial cells [49]. MiRNAs have a profound impact on the regulation of allergic inflammation and are expected to become biomarkers for allergic diseases, such as asthma, as well as important therapeutic targets in the future. MiRNAs with a profound impact on the regulation of allergic inflammation are expected to be biomarkers for allergic diseases, such as asthma, and may likewise be a valuable therapeutic target in the future.

2. MicroRNAs and ILC2s

Derived from typical lymph-like progenitor cells in the bone marrow and expressed in tissues, such as the lung, ILC2s are considered the innate counterpart of Th2 cells and have a collaborative role in the pathogenesis of allergic diseases. Belonging to the innate immune system, ILC2s do not articulate rearranged specific antigen receptors, but their activation is mainly controlled by epithelial cytokines, such as TSLP, IL-25, and IL-33. Among these, IL-33 and IL-25 are potent activators of ILC2. ILC2s can be characterized by the generation of type 2 cytokines (IL-13, IL-4, and IL-5) and the expression of GATA-3 transcription factors. Located strategically in the airway mucosa, ILC2 can be critical for patrolling the airway, enrolling other immune system cells, and activating resident cells in response to pathogenic injury or tissue damage [50]. Consequently, ILC2s have been studied for their development, proliferation, and expression. The study of the underlying mechanisms of ILC2 is of current interest; however, extensive research on ILC2s has in recent years yielded the conclusion that it is ILC2s that act as the significant secretory drivers of allergic inflammation and contribute significantly to airway disease models [51]. Therefore, the following section will describe the effects of various miRNAs, including miR-155, miR-1146, and miR-206, on ILC2s, as an increasing amount of research has been devoted to ILC2s.

2.1 MiR-155

There is no doubt that the development of the immune system, the maturation and differentiation of immune cells, and the stability of their function are inseparable from the involvement of miR-155, which participates in the modulation of the Th2 immune response in eosinophilic airway inflammation. Analyzing the nasal mucosa and peripheral blood collected from allergic rhinitis (AR) patients, Zhu revealed that the miR-155, IL-25, and IL-33 in nasal mucosa exceeded those in controls, as did ILC2s in blood, all of which had statistical significance. Later, a mouse model of LAR was built to validate this idea and concluded that miR-155 was indispensable in the proliferation and activation of ILC2s induced by IL-33 [52]. In other words, overexpression of miR-155 facilitates the overexpression of ILC2s, triggering many type 2 cytokines and a series of allergic phenotypes. The same conclusion came from Wan’s study of AR mouse models, where miR-155 not only promoted Th2 differentiation but also increased the number of ILC2s [53]. With the mouse model of asthma, however, Martin concluded that miR-155 could protect ILC2 from apoptosis in a way that boosted the type 2 immune response, given the small effect of miR-155 on ILC2 cell proliferation as well as cytokines [54]. Indeed, they have mutual recognition of miR-155 as required for ILC2s to respond to IL-33 amplification [55]. The proliferation in the absence of miR-155 can be complex for those ILC2s, albeit through intrinsic mechanisms that need further investigation. Nevertheless, one thing is sure, miR-155 is instrumental in the proliferation, activation, and functional stability of ILC2s.

2.2 MiR-146

The anti-inflammatory properties of MiR-146a can be seen in human bronchial epithelial cells during rhinovirus infection and allergic inflammation and in the airways of mice [56]. Targeting miR-146 may be a novel strategy for the treatment of allergic asthma. In mouse models of asthma, treatment of IL-33-activated ILC2s with miR-146a decreased the ability of ILC2s to secrete cytokines, whereas no effects were observed in IL-25-activated ILC2s. Meanwhile, the ability of ILC2 to secrete IL-13 and IL-5 was increased after miR-146a inhibitor treatment, and the results after miR-146 administration showed no significant change in IL-33 and IL-25 expression levels in mice [57, 58]. Thus, miR-146a may reduce the airway inflammatory response in asthma by blocking the IL-33 pathway, by inhibiting IRAK1 and TRAF6, downstream molecules of ST2 signal pathway, to negatively regulate IL-33/ST2-activated ILC2 to inhibit asthma [59]. But a pathway other than IL-25 cannot be excluded. Consequently, we conclude that miR-146a could be one of the most effective therapeutic options for the anti-inflammatory treatment of asthma.

2.3 Other ILC2s

According to Zhang, T2-type asthmatics showed lower airway epithelial miR-206 than normal subjects, including higher levels in type 2 hyper asthmatics than in type 2 hypo asthmatics. As a result, an animal model is built to suggest that higher miR-206 in type 2 hyper asthma can reduce CD39, an ATP degrading ectonucleotidase and a target of miR-206, whose accumulation exacerbates asthma [60]. Many cells can secrete EVs extracted from the supernatant of human mast cells (MC). Their miRNA profiling shows that miR103a-3p is markedly up-regulated and enhances IL-5 production by ILC2s after coculture with human ILC2s [61] through methylation of GATA3 arginine residues. While ICAM-1 appears to be essential for ILC2 development and function, it has been found in AR patients and animal models that overexpression of miR-150-5p can decrease ICAM-1 expression, whereas a decrease in ICAM-1 can led to a downregulation of GATA3 levels, which can, in turn, inhibit the function of ILC2s from alleviating allergic symptoms [62]. The results of an in vivo study in a mouse model of AR showed that miR-375 enhanced the function of ILC2s by regulating TSLP [63], providing a potential therapeutic target for AR. The miR-17 ∼ 92 gene cluster Th2 secretes related cytokines, of which miR-19a takes a significant role. By constructing a mouse model of asthma, miR-19a promoted the activation and proliferation of ILC2s, thereby increasing the secretion of IL-13 and IL-5 [51]. Roberts’ study showed a biased development of ILC2s progenitors in the bone marrow of mir142−/− mice compared to wild-type mice, with a dysregulated proliferative effect of ILC2s [64]. These findings indicate that miR-142 maintains the homeostasis of ILC2s in tissues, similar to miR-155 and miR-19. With the increasing prominence of ILC2s in allergic diseases as a top research priority related to the pathogenesis of asthma to date, it is believed that further studies will undoubtedly identify additional relationships between miRNAs and ILC2s.

3. The relationship between microRNAs, ILC2s, and Th2 cells

Asthma is a complex disease engaged by multiple mechanisms on a nonhomogeneous basis. Asthma has variable conditions in terms of clinical presentation (phenotype) and different pathophysiological mechanisms (endotype) [65]. An imbalance in the Th1/Th2 ratio and hypersecretion of Th2 cytokines are currently thought pivotal in the pathogenesis of allergic asthma. Cells that produce Th2-type cytokines include type 2 helper T cells (Th2), follicular helper T cells (TFH), basophils, mast cells, and type 2 innate lymphoid-like cells (ILC2s). ILC2s and Th2 cells can induce type 2 immunopathology by releasing type 2 effector cytokines [66]. As a result, ILC2s offer a primary source of early intrinsic cells driving the classical type 2 cytokines in eosinophilic inflammation. Participating as regulators of immunity by coordinating multiple target genes in multiple cells, MiRNAs can modulate Th2 cell differentiation, Th1/Th2 balance, and promote type 2 immune responses, and thus participate in the development and progression of asthma. ILC2s are critical initiators of allergic inflammation. They all participate in asthma onset and development. Many miRNAs mentioned above are involved in Th2 cell responses and ILC2s reactions, contributing to more or less the development of allergic diseases. Priti, who compared the miRNAs transcriptomes of ILC2s and Th2 cells in lung tissue, concluded that miRNAs expression is necessary to maintain ILC2 homeostasis in vivo, which can be mediated by participating in the regulation of overlapping but not identical target genes of innate and adaptive immune cells to achieve certain expected biological outcomes that can contribute to the development of allergic disease [51]. Therefore, we might also consider that specific miRNAs affect both Th2 and ILC2s through common pathways, exacerbating or contributing to certain allergic diseases, such as asthma. A common link between MicroRNAs, ILC2s, and Th2 remains to be further investigated.