Wnt Signaling in Allergic Asthma and Pediatric Lung Diseases

Structured abstract

Purpose of review:

To provide an update on the current understanding of the role of Wnt signaling in pediatric allergic asthma and other pediatric lung diseases.

Recent findings:

The Wnt signaling pathway is critical for normal lung development. Genetic and epigenetic human studies indicate a link between Wnt signaling and the development and severity of asthma in children. Mechanistic studies using animal models of allergic asthma demonstrate a key role for Wnt signaling in allergic airway inflammation and remodeling. More recently, data on BPD pathogenesis points to the Wnt signaling pathway as an important regulator.

Summary:

Current data indicates that the Wnt signaling pathway is an important mediator in allergic asthma and BPD pathogenesis. Further studies are needed to characterize the roles of individual Wnt signals in childhood disease, and to identify potential novel therapeutic targets to slow or prevent disease processes.

Introduction

Respiratory diseases account for the majority of pediatric hospitalizations in the United States, and remain the major cause of childhood morbidity and mortality worldwide [1,2]. Amongst them, asthma is the most common chronic condition amid children, and places an enormous burden on families, the healthcare system, and society as a whole [3]. Other less common lung diseases such as cystic fibrosis, bronchopulmonary dysplasia (BPD), and interstitial lung diseases are becoming more prevalent with advances in diagnostic technologies and improved survival. Understanding the pathogenesis of these diseases is of great importance as we seek more effective treatments.

In efforts to elucidate the molecular pathogenesis of childhood respiratory diseases, researchers have gained insight from the signaling pathways involved in early embryonic development. Remarkably, signals and mechanisms that are active in organogenesis are later involved in post-natal, childhood, and adult tissue homeostasis and response to injury [4]. The Wingless/integrase-1 (Wnt) signaling pathway, best known for its roles in human carcinogenesis and embryonic development, is one of the key developmental signaling pathways and a major area of interest in pulmonary pathology [5]. In this review, we will focus on recent studies describing the role of Wnt signaling in lung development, allergic asthma, and BPD.

Overview of Wnt signaling

The Wnt signaling pathway is a cellular communication system that is essential for stem cell renewal, proliferation, and differentiation. It is active during embryogenesis, homeostasis of mature tissues, and repair of injured tissues. Initially identified in the context of fruit fly morphogenesis and tumorigenesis in mice, it was later established that the Wnt pathway is evolutionarily conserved and that Wnt homologs are critical for normal vertebrate development. There are 19 different Wnt proteins encoded by the mammalian genome, and they have emerged as major growth factors in human development and disease [6].

Wnt signaling is classified into canonical and non-canonical signaling. Canonical Wnt signaling, which is the best characterized to date, relies on downstream activation of β-catenin which exerts influence on gene expression in the nucleus. In the absence of Wnt protein signaling, β-catenin is maintained at low levels in the cytoplasm by a destruction complex. This complex, composed of axin, adenomatous polyposis coli, casein kinase I, and glycogen synthase kinase-3β, phosphorylates and ubiquitinates β-catenin, leading to proteasomal degradation. Wnt signals, which are palmitoylated and secreted into the extracellular space, bind to cell surface Frizzled (Fzd) and low density lipoprotein receptor-related proteins 5 and 6 (LRP 5/6). This interaction initiates a signaling cascade that inactivates the destruction complex and allows accumulation of cytosolic β-catenin. β-catenin translocates into the nucleus, interacts with the transcription factors T-cell factor/lymphoid enhancer factor-1 (TCF/LEF), and induces gene transcription. Wnt target genes are generally involved in cell-cycle progression, stem cell function, and feedback regulation of the pathway, which vary depending on the cell and tissue type [7,8]. (Figure 2). The non-canonical pathways operate independently of β-catenin, and are involved in planar cell polarity, cell movement and calcium/calmodulin-dependent protein kinase II signaling [8,9].

Figure 2:

Overview of canonical Wnt/β-catenin signaling. The left panel shows how β-catenin is degraded by the β-catenin destruction complex in the absence of Wnt ligands. The right panel shows how active Wnt signaling inactivates the β-catenin destruction complex and allows β-catenin to migrate to the nucleus and interact with TCF/LEF, promoting transcription of Wnt target genes. Created with BioRender.com.

Wnt signaling and lung development

Lung development is divided into five stages: embryonic, pseudoglandular, canalicular, saccular, and alveolar. These stages require highly coordinated cell proliferation and differentiation to ensure appropriate branching, crosstalk between the mesenchyme and endoderm, and maturation of the over 40 different known respiratory cell types [10]. The Wnt signaling pathway, with its instrumental control over the cell cycle and stem cell development, plays an important role in each phase of lung development (Figure 1) [11]. Starting during the embryonic stage, Wnt2+ cardiopulmonary mesoderm progenitor cells are responsible for generating the mesoderm lineages that give rise to the cardiac inflow tract and lung [12]. Wnt2/2b and β-catenin promote specification of foregut cells into Nkx2.1+ lung endodermal cells, and mice that lack Wnt2/2b develop lung agenesis [13]. Wnt2 and Wnt7b promote mesenchymal smooth muscle cell differentiation which are required for airway development [14].

Figure 1:

Wnt/β-catenin signaling in normal lung development. Created with BioRender.com.

In the pseudoglandular stage, during which the conducting airways are formed, Wnt/β-catenin signaling is active at the start of branching morphogenesis. Disruption in R-spondin 2 (RSPO2), a potentiator of β-catenin signaling, leads to laryngeal-cartilage malformations and lung hypoplasia in the mouse model, and recessive human RSPO2 mutations have been reported to cause a lethal form of tetra-amelia syndrome characterized by absent limbs and lung agenesis [15,16]. Wnt5a is highly expressed in mesenchyme and regulates elongation of the trachea, and through its influence on other signaling pathways, controls distal lung morphogenesis [17,18]. Wnt7b promotes mesenchymal proliferation and pulmonary angiogenesis, and mice deficient in this gene develop lung hypoplasia [19]. Immunolocalization studies demonstrated that secreted frizzled related protein-1 (SFRP-1) is highly expressed in the distal airway epithelium during the late pseudoglandular stage, and genetic ablation of Sfrp-1 in mice impaired distal lung development [20]. In the canalicular and saccular stages, during which there is development of the airspaces required for gas exchange, β-catenin is required for distal airway formation and regulates differentiation of lung progenitors [21–23]. During the alveolar stage, Wnt-responsive type 2 alveolar cells promote alveologenesis, and inhibition of Wnt signaling promotes differentiation into alveolar type 1 cells [24].

Wnt and allergic asthma

Genetic studies

Understanding the genes and cell signals that orchestrate normal lung development can provide insight into disease processes. In an elegant study by Sharma et al, variants in genes that are differentially expressed during in utero airway development were examined in patients with asthma. Genetic association analyses of lung function for variants in these genes was performed in two childhood asthma cohorts that included 986 parent-child trios. Single nucleotide polymorphisms (SNPs) in WIF1 and WISP1 were associated with impaired lung function in both cohorts, indicating a role for these Wnt signals in the pathobiology of asthma [25]. Another study using a pediatric asthma cohort from China showed a similar association between genetic variations in the WISP1 gene and lung function [26]. Gene set enrichment analysis demonstrated enrichment of the Wnt signaling pathway in patients with asthma, and association studies identified an asthma susceptibility locus near FZD6, a receptor for Wnts [27]. Numerous studies have confirmed an association between variants in FAM13A, which encodes a protein that promotes β-catenin degradation, in asthma and COPD in non-smokers [28,29].

Epigenetics

One of the major limitations of genome association studies, and perhaps the reason that investigators have not yet detected any single genetic variant that can explain a significant proportion of the heritability of asthma, is that genome association studies do not account for the environmental factors that contribute to asthma pathogenesis. Epigenetic studies aim to identify gene-environment interactions by measuring molecular DNA modifications that alter gene expression. One study demonstrated that patients with asthma who had clinical remission had differential methylation in the gene TCF7L2, Wnt transcription factor [30]. In another interesting study examining maternal psychological stress and the development of wheeze later in their offspring, the authors described increased transcription of β-catenin and Axin2 in children of highly stressed mothers, and elevated β-catenin expression at birth was associated with increased risk of the child developing persistent wheeze later in life [31].

Allergic inflammation

In addition to genomic and epigenetic approaches, mechanistic studies are key to understanding the function of the Wnt signaling pathway. Most mechanistic studies in asthma focus on allergic asthma, the most common and most understood phenotype [32,33]. Allergic asthma typically starts in childhood and tends to coexist with allergic rhinitis, food allergies, and atopic dermatitis [34]. Sensitization to aeroallergens leads to eosinophilic airway inflammation, manifesting in lower airway bronchoconstriction and mucus production. The immunopathogenesis is characterized by polarization of helper T cells into the type 2 (Th2) subtype. Th2 cells promote production of cytokines IL-4, IL-13 and IL-5, which cause IgE class switching and eosinophil activation, leading to allergic airway inflammation. New biologic therapies for children and adults with severe asthma target components of Th2 driven inflammation and can reduce exacerbations and glucocorticoid dependence [35].

In the quest to understand the exact mechanism by which individuals with allergic asthma have a predilection towards Th2 inflammation, the Wnt signaling pathway is an area of interest. In fact, one of the key transcription factors of this pathway is T cell factor (TCF), which plays a critical role in T cell fate specification and is required for T cell development in the thymus [36,37]. Using an in vivo house dust mite asthma model, Trischler et al demonstrated that genetic ablation of Wnt10b led to increased Th2 polarization of splenic T cells. Accordingly, Wnt10b^−/− mice exhibited increased eosinophilic airway inflammation, and higher concentrations of IL-4 and IL-13 in bronchoalveolar lavage [38]. In two key studies, Reuter et al established that dendritic cell dependent T cell activation is impaired by inhibiting degradation of β-catenin, and that promoting Wnt signaling leads to attenuated airway inflammation in an animal model [39,40]. A recent study revealed a new subset of regulatory T cells that express Notch4 and produces Th2 and Th17 cytokines, thereby promoting allergic asthma. This promotion of allergic inflammation and dampening of immunoregulatory function occurred through Wnt and Hippo pathway dependent mechanisms [41].

Studies exploring the roles of negative regulators of the Wnt/β-catenin pathway have also elucidated important findings in asthma. Using bronchial epithelium samples from patients with asthma, Hachim et al observed that a Th2 high phenotype was associated with upregulation of Wnt inhibitory modulators [42]. Consistent with this, our group demonstrated that SFRP-1, an endogenous protein that binds to Frizzled receptors and inhibits Wnt ligand binding, potentiates allergic asthma in a house dust mite mouse model, and genetic ablation of Sfrp-1 led to reduced alternatively activated alveolar macrophages compared to wild type mice [43]. Dickkopf-1 (DKK-1), a Wnt antagonist, promoted polarization of T cells into Th2 cells, and its functional inhibition in a house dust mite asthma model was protective [44].

Airway remodeling

After repeated cycles of exposure to aeroallergens, inflammation, bronchoconstriction, and resolution, structural changes in the airways develop and lead to permanent narrowing and airflow limitation [45]. These changes are characterized histologically by airway wall thickening, subepithelial fibrosis, myofibroblast hyperplasia, increased airway smooth muscle (ASM) mass, and mucus metaplasia [46,47]. The age of onset of airway remodeling is not known, but is has been detected in school age children with difficult-to-treat asthma and wheezy preschool children [48–50]. The mechanism by which airway remodeling occurs, and to what extent new biologic therapies can reverse it, is not fully understood [51].

Wnt/β-catenin signaling is required for development and differentiation of airway epithelial and smooth muscle cells, and thus has a biologically plausible role in structural airway changes in asthma [52]. Using a mouse model of chronic allergic asthma designed to induce remodeling features, Kwak et al established that β-catenin is expressed in the remodeled airways. Inhibition of β-catenin expression during the allergen challenge attenuated subepithelial fibrosis, collagen content, and expression of the profibrotic cytokine TGF-β [53]. A more recent study explored the therapeutic potential of mesenchymal stem cells (MSCs), which are pluripotent stem cells with immunoregulatory functions. Sensitized rats that received treatment with bone marrow-derived MSCs had reduced airway remodeling and downregulation of the Wnt signaling pathway proteins [54]. Consistent with these studies, suppression of a Wnt antagonist, secreted frizzled-related protein 4 (SFRP-4), led to increased airway remodeling [55]. Additionally, in vitro studies link noncanonical Wnt5a with TGF-β induced extracellular matrix deposition by ASM cells and promotion of actin polymerization required for airway contraction [56,57]. Taken together, these results emphasize the importance of both canonical and noncanonical Wnt signaling in airway remodeling, but further work is required to reconcile the somewhat divergent roles of β-catenin in allergic inflammation and later structural airway changes. It is clear though that carefully orchestrated expression of Wnt molecules in particular cell types is required to regulate the precise activation of the pathway. A summary of the role of Wnt signals in the pathogenesis of allergic asthma is provided in Figure 3.

Figure 3:

The role of individual Wnt signals in the pathogenesis of allergic asthma. Created with BioRender.com.

Wnt and Bronchopulmonary Dysplasia

BPD is the most common chronic lung disease in infancy and the most common complication of prematurity [58]. While advances in medical care have led to improved survival of neonates born at earlier gestational age, the prevalence of BPD is rising in many high-income countries [59]. In order to reduce the incidence of BPD and provide optimal long-term care for children born prematurely, a better understanding of the mechanisms that drive multifactorial lung injury in the immature and growing lung is essential. Human mechanistic studies in BPD are limited given the risks associated with invasive pulmonary sampling in infants, but based on currently available data, the Wnt signaling pathway is emerging as a potentially key pathway in the pathogenesis of the disease.

Twin studies suggest that genetic factors are involved to some degree in the development of BPD [60]. Li et al performed exome sequencing on DNA isolated from neonatal blood spots and identified that genes involved in the regulation of the Wnt signaling pathway were highly enriched in samples from individuals with BPD compared to those without BPD [61]. Histological studies using lung tissue from infants who died from BPD demonstrated increased immunofluorescent β-catenin staining in the thickened alveolar interstitium [62]. These findings were expanded by Sucre et al who described nuclear localization of β-catenin in the alveolar septa of mid-gestation fetal lungs and lungs with BPD, but not in the lungs of full term infants [63]. In vitro studies using 3-dimensional organotypic cocultures of human fetal alveolar type II cells and pulmonary fibroblasts exposed to hyperoxic conditions demonstrated increased Wnt2b, Wnt5a, Wnt9a, and Wnt16 expression, and ex vivo experiments on hyperoxia-exposed precision-cut lung slices from mouse pups confirmed that mesenchymal expression of Wnt5A drives impaired alveolarization in response to hyperoxia [64]. Consistent with this, a study investigating how fibroblasts exposed to hyperoxia are unable to support alveolar epithelial differentiation in organoid culture revealed that these fibroblasts have reduced Wnt signaling. Activation of canonical Wnt signaling in these cells restored organoid formation and growth after exposure to hyperoxia [65]. In summary, initial studies indicate aberrant Wnt signaling in BPD promotes lung injury, but further studies are needed to determine its exact role in this disease.

Wnt and congenital pulmonary abnormalities

At this time, very little is known about the role of Wnt signaling in human congenital malformations that affect the respiratory system. A human homozygous Wnt4 mutation causes a lethal autosomal recessive disorder called SERKAL (SEx Reversal, dysgenesis of Kidneys, Adrenals, and Lungs) syndrome, highlighting the importance of Wnt4 in organogenesis [66]. Murine studies indicate a link between congenital cystic lung disease, mesenchyme-specific TGF-β signaling, and downregulation of the canonical Wnt signaling pathway [67]. Advances in genomic technology and precision medicine will likely provide more data on the role of Wnt signaling in other less common pediatric lung diseases.

Conclusions

The Wnt/β-catenin signaling pathway is essential during lung development and plays an important role in pediatric asthma and BPD. Future studies are needed to determine if specific proteins in this pathway can be targeted as points of novel therapeutic intervention to prevent lung damage in these common diseases.

Key Points.

• The Wnt/β-catenin signaling pathway is essential for normal lung development.

• Genetic and epigenetic human studies indicate that Wnt signaling impacts the development and severity of asthma in children.

• Studies in animal models of asthma demonstrate a key role for Wnt signaling in allergic inflammation and airway remodeling.

• Emerging data on the pathogenesis of BPD points to the Wnt signaling pathway as an important regulator.

• Further studies are needed to determine potential therapeutic targets within the Wnt signaling pathway that may impact the evolution of allergic asthma and BPD in children.