Lung disease manifestations in Down syndrome

Abstract

Down syndrome (DS) is one of the most prevalent chromosomal abnormalities worldwide, affecting 1 in 700 live births. Although multiple organ systems are affected by the chromosomal defects, respiratory failure and lung disease are the leading causes of morbidity and mortality observed in DS. Manifestations of DS in the respiratory system encompass the entire lung starting from the nasopharynx to the trachea/upper airways to the lower airways and alveolar spaces, as well as vascular and lymphatic defects. Most of our knowledge on respiratory illness in persons with DS arises from pediatric studies; however, many of these disorders present early in infancy, supporting developmental mechanisms. In this review, we will focus on the different lung phenotypes in DS, as well as the genetic and molecular pathways that may be contributing to these complications during development.

INTRODUCTION

Down syndrome (DS) is the most prevalent chromosomal abnormality worldwide, with an incidence of ∼1 in 700 live births (1, 2). There are three genetic variations defining Down syndrome: 1) trisomy 21, which accounts for ∼95% of all DS cases and is the result of an additional partial or complete copy of human chromosome 21 (Hsa21); 2) mosaic Down syndrome, which is quite rare and results from a subset of cells having an extra copy of Hsa21; and 3) translocation Down syndrome, where an extra portion of Hsa21 (either inherited or sporadic) is translocated onto another chromosome. These different genetic variations are believed to contribute to the varied degrees of phenotypic severity observed among individuals with DS (3). Multiple organ systems are affected by these chromosomal defects, including but not limited to craniofacial abnormalities, gastrointestinal issues, cognitive deficits, autoimmune disorders, and hematological abnormalities. Although each of these impediments is of significance, the high incidence of prolonged hospitalizations in children with DS is due to congenital cardiac defects and respiratory issues, thus causing around 75% of the mortality observed in DS (2). Approximately 40% of the newborns with DS present with some version of congenital heart defect, including atrioventricular cushion defects and ventricular septal defects (4). More than 54% of the hospitalizations of persons with DS are due to lung disease, with respiratory complications being the most common cause of hospital admissions in children with DS of age 3 yr or younger (5). Most of our knowledge on respiratory illness in persons with DS arises from pediatric studies, with little data from the adult population or prenatal cohorts, yet even in the adult population with DS, lung disease remains a leading cause of morbidity and mortality (2, 6). Many of these disorders present early in infancy, supporting developmental mechanisms. However, a better understanding of respiratory failure and disease in DS is needed. Manifestations of DS in the respiratory system start from the nasopharynx to the trachea/upper airways to the lower airways and alveolar spaces in addition to vascular and lymphatic defects. The diversity and complexity of these aberrations remain poorly understood, but pulmonary complications associated with DS encompass pulmonary vascular disease, sleep-disordered breathing, interstitial lung disease, airway abnormalities, autoimmune conditions, and infections due to impaired immunity (7). Although animal models and in vitro systems have proven to be critical in understanding many of the neurodegenerative and hematological anomalies observed in DS, the intricacies associated with DS lung disease prove to be challenging to replicate. Many of these manifestations are detailed herein and summarized in Table 1 and Fig. 1, followed by a brief description of the models currently used and their limitations when studying DS lung disease.

Table 1.

Respiratory manifestations in Down syndrome

Area/Region	Manifestations	Possible Mechanisms	References
Upper airways	Macroglossia	Hypermethylation of HOXB cluster Hypermethylation of HOXB cluster Hypermethylation of HOXB cluster Hypermethylation of HOXB cluster Hypermethylation of HOXB cluster Hypermethylation of HOXB cluster ↑ PCNT	(8)
	Adenotonsillar hypertrophy		(8)
	Laryngomalacia		(8)
	Narrow trachea/tracheomalacia		(2, 9, 10)
	Tracheal bronchus		(9)
	Obstruction		(2, 8, 11)
	Ciliopathies/increased mucus production		(12, 13)
Lower airways	Recurrent infection Poor airway clearance	Cytokine/chemokine storm ↑ IFN pathway ↑ PCNT	(14, 15, 16, 17, 18, 19) (14, 15, 16, 17, 18, 19) (12, 13)
Alveolar space	Pulmonary hypoplasia Subpleural cysts	↑ COL6A1 ↑ COL6A1	(20, 21, 22) (23)
Vascular	Pulmonary hypertension Pulmonary hemosiderosis Pulmonary capillaritis	↑ COL18A1, APP, TIMP3, COL4A3, RCAN1, DSCR1 ↓Clotting factors (F2, F5, F7, F12) ↑ COL18A1, APP, TIMP3, COL4A3, RCAN1, DSCR1 ↓Clotting factors (F2, F5, F7, F12) ↑ COL18A1, APP, TIMP3, COL4A3, RCAN1, DSCR1 ↓Clotting factors (F2, F5, F7, F12)	(4, 24, 25) (21) (26, 27) (21) (28) (21)
Lymphatics	Lymphangiectasia	↑ SHH, VEGF-A, PDG-B ↓FOXC2 ↑IFN pathways	(12, 29) (21, 29) (21, 29)

Disease manifestations are listed by region of the lung affected.

Figure 1.

Cartoon summarizing the mechanisms underlying lung disease in Down syndrome. This cartoon is demonstrating an individual with Down syndrome. It is summarizing the possible upper airway, lower airway, alveolar space, and endothelial anomalies these individuals may experience. Furthermore, it is suggesting the possible mechanisms that may be contributing to these anomalies. For more information regarding the pathologies and the potential mechanisms, refer to Table 1 and the associated references. [Created with BioRender.com “Macro to Micro View of the Lungs” template and published with permission.]

UPPER AIRWAY ANOMALIES

Upper airway defects in individuals with DS often present as a smaller and narrower trachea, requiring endotracheal tubes at least two sizes smaller than their age-matched non-DS counterparts (10), in addition to a tracheal bronchus associated with persistent or recurrent pneumonia (9). In addition, individuals with DS present with reduced upper airway muscle tone or dysphagia and bronchomalacia (30). Narrowing of the upper respiratory tract often contributes to recurrent pneumonia, and airway malacia leads to obstruction in >50% of children (2). Many children with DS also present with laryngotracheal abnormalities, which include tracheal stenosis due to the formation of complete tracheal rings, and laryngomalacia, which is a softening of the tissues surrounding the larynx resulting in their floppiness. In addition, a large subset of these children experience macroglossia, in which the tongue may have the potential to extend past the incisors, and adenotonsillar hyperplasia (8). Either one of these irregularities, or a combination, may result in the most common respiratory disorder observed in DS, obstructive sleep apnea, which affects up to 75% of the DS population (8, 11). Although it is expected that there is a 50% increased expression of the genes located on Hsa21, given the additional chromosome number, it is interesting to note that genome-wide transcriptional changes are also observed, perhaps due to the overexpression of chromosome modifiers located on Hsa21 (31, 32). There is no evidence of one or a set of specific genes responsible for these anomalies. Therefore, many of the upper airway anomalies may rather be a result of epigenetic modifications such as hypermethylation of the HOXB cluster (33), which has been shown to play a critical role in controlling airway patterning (34).

LOWER AIRWAY/ALVEOLAR ANOMALIES

Postnatal DS lungs are often observed as being hypoplastic, a condition defined by an incomplete development of the lung due to decreased lung cells, airways, alveoli, and, in result, organ size (35). DS lungs generally present with a porous phenotype resulting from the reduced number of alveoli (between 58% and 83% of the expected number) and enlarged alveolar airspaces (36) and could also have up to a 25% decrease in branch generation number (22). As the bronchial tree is established during the pseudoglandular stage of lung development, this finding suggests early prenatal interference in lung formation. These lungs also present with subpleural cysts, a phenomenon rarely witnessed in individuals not having DS, whereas the prevalence in DS is around 36% (Fig. 2, A and B). Although the etiology of these cysts is poorly understood, they are associated with pulmonary hypoplasia and are thought to represent deficient postnatal alveolarization (Fig. 2C) (23). As many of these conditions remain constant into adulthood as opposed to becoming exacerbated, the pulmonary complications observed are likely due to developmental insufficiency (or perturbations). This has been confirmed by our recent study, where we demonstrate that >70% of prenatal lungs analyzed present histological abnormalities, with >60% at 16+-wk gestation displaying airway dilatation (21). We also demonstrated that these lungs present with defects in extracellular matrix components, which are known to play a critical supporting role/environment for epithelial cells to proliferate, migrate, and differentiate (21). One of the dysregulated genes is COL6A1 (Collagen Type VI Alpha 1 Chain), which is upregulated in T21 lungs, as it is localized on Hsa21. Previous studies have shown that deletion of Col6a1 in the mouse results in increased branching (37); therefore, there is a possibility that the increased expression found in individuals with DS may contribute to the hypoplastic phenomena reported.

Figure 2.

Lung pathology in Down syndrome. A and B: subpleural cystic dilatation of alveolar spaces (arrows) are characteristic, visualized on both gross and microscopic examination of the lung. C: pulmonary hypoplasia in an 8-mo-old infant with congenital heart disease is visualized on lung biopsy by the markedly enlarged alveolar spaces, compare with alveolar spaces in B and size of the airways (*). D: a double-capillary network in the alveolar septa (arrowheads) of a term infant with trisomy 21 (left), compared with normal term alveolar septa (right). E: a 2.5-mo-old with complex congenital heart disease and severe pulmonary hypertension has occlusive wall thickening in a pulmonary artery (a). F: pulmonary hemosiderosis (brown pigment in alveolar macrophages) and acute hemorrhage in a 3-yr-old who presented with anemia.

In the lower respiratory tract, viral and bacterial infections are the most common cause for hospital admissions in children with DS and combined with congenital heart disease are the leading cause of morbidity and mortality (38). These individuals present with ciliopathies, due to aberrant pericentrin (PCNT) expression (12), in addition to increased mucus production (13), establishing a prime environment for lower respiratory tract infections. Respiratory syncytial virus (RSV) is the most common pathogen in these infections. Children with DS under the age of 2 are 6.8-fold more likely to be hospitalized for acute lower respiratory tract infections from RSV than children without DS (14). Furthermore, a number of immune defects detected in DS could result in an increased incidence of respiratory diseases and infections. These include reduced B- and T-lymphocytes, perhaps due to increased apoptosis (16), altered immunoglobulin expression, and decreased monocytes (15, 18). Children with DS also exhibit baseline cytokine and chemokine storms with both a pro- (IL-2, IL-6) and an anti-inflammatory (IL-10, IL-1ra) phenotype, likely increasing these individuals’ susceptibility to autoimmunity and sepsis, which may be a result of overactivation of the interferon (IFN) pathway (17, 19).

Hyperactive IFN signaling is well established in DS, likely due to the fact that four of the six IFN receptors are encoded on Hsa21, namely, IFNAR1, IFNAR2, IFNGR2, and IL10RB, ultimately suggesting an important role in DS pathophysiology (39, 40). In addition to the increased expression of these receptors in DS cells, it was further demonstrated that IFN-related factors (including ligands and IFN-activated transcription factors) are differentially expressed (21, 29). Furthermore, DS cells treated with IFN ligands demonstrate an important increase in IFN pathway activity as compared with non-DS cells, suggesting a positive feedback loop (39). In addition to demonstrating hyperactive IFN signaling in a diverse immune cell population, transcriptome analysis on DS skin fibroblasts shows elevated IFN expression in many nonimmune cell types (41, 42). Many groups have also shown that the IFN pathway plays a significant role in the neurodevelopment of individuals with DS (43). Furthermore, the IFN pathway regulates differentiation, proliferation, and apoptosis in a number of different cell types and organs (44, 45, 46), all of which are important for lung organogenesis. In addition, our studies have recently demonstrated an upregulation of type I IFN signaling in human prenatal T21 lungs (21). Therefore, the mechanisms by which aberrant IFN signaling contributes to the abnormal lung phenotype observed in DS require further investigation.

Type I IFN signaling is the first line of defense in viral infections, blocking early viral replication and spread. This poses a very interesting question as to whether individuals with DS are more or less susceptible to the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. A recent study demonstrated that primary human airway epithelial (pHAE) cultures infected with COVID-19 lacked a type I IFN response. However, when the cells were pretreated or posttreated with type I or type III IFN ligands to activate the IFN response, it promoted restriction of viral replication as well as reduced viral levels (50). Alternatively, it has been demonstrated that increased type I IFN expression in response to viral infections increases the risk of deadly pulmonary bacterial infections, to which individuals with DS are more susceptible. Furthermore, it should be noted that transmembrane serine protease 2 (TMPRSS2), which encodes a serine protease and facilitates the entry of SARS-CoV-2 into the cell, is located on Hsa21 and is upregulated in the lung of individuals with DS. Therefore, it is not unexpected that recent studies demonstrated that adults with DS are at an increased risk of severe disease (pneumonia), and infants with DS can present with severe multisystem inflammatory syndrome I children (MIS-C) after COVID-19 infection (48, 49). The mechanisms leading to immune defects in DS, and their contribution to an increased risk of infection, remain poorly understood.

This further raises the question as to how these individuals would respond to a COVID-19 vaccine. The immune dysregulations observed in individuals with DS compromise the establishment of protective immunity initiated by vaccination. It has been demonstrated on several occasions that vaccine efficacy in DS is diminished due to poor immune response and may require more frequent booster immunizations to maintain the immunity already established (50, 51). Unfortunately, to date, there is no clear time frame as to how long COVID-19 vaccines offer protection to those immunized. The Centers for Disease Control and Prevention is currently recommending that immunocompromised individuals receive an additional dose of the mRNA COVID-19 vaccines. In addition to compromised immunity contributing to the complications observed in the lower airways/alveoli of DS lungs, pulmonary vascular diseases also hinder alveolar development.

ENDOTHELIAL ANOMALIES

Surrounding the alveoli is a unique vasculature, displaying an immature double capillary network system, something not typically seen in other lung conditions (Fig. 2D) (20). The persistence of a double-capillary network system is representative of the saccular stage of development (20), as opposed to a single-capillary network that is normally observed postnatally, during the alveolar stage. This condition results in more space between the capillaries as well as an increased incidence of intrapulmonary anastomoses (2). In addition, changes in endothelial permeability have been noted, resulting in embolism and edema of the lung that are thought to derive from potassium and calcium channel disturbances (2). Several genes located on Hsa21 have been shown to be involved in DS pathogenesis, such as regulator of calcineurin 1 (RCAN1), Down syndrome critical region gene 1 (DSCR1), DS cell adhesion molecule (DSCAM), synaptojanin1 (SYNJ1), and dual-specificity tyrosine phosphorylation-regulated kinase (DYRK1A). Although, many of these genes are correlated to cognitive issues, they may also play a potential role in the respiratory complications observed in individuals with DS, such as pulmonary arterial hypertension. For example, increased activation of the calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) signaling pathway, which is activated by increased RCAN1/DSCR1, is critical to the downstream pathway store-operated Ca²⁺ entry (SOCE) (52). Although a clear connection between SOCE and regulation of vascular smooth muscle tone and the activation of angiogenesis has been shown (53), the influx of calcineurin may ultimately have an effect on the airway smooth muscle cells (ASMCs) and airway remodeling in a DS lung. Calcineurin, which is a Ca²⁺-calmodulin-dependent phosphorylase, activates NFAT, which may then modulate the gene expression of factors shown to influence ASMC hypertrophy and proliferation (54). The CaN/NFAT pathway is known to regulate lung maturation by modulating the expression of the different surfactant genes (55, 56).

Furthermore, an upregulation of antiangiogenic genes encoded on Hsa21 within the developing human DS lung, such as COL18A1 (endostatin, ES), amyloid beta precursor protein (APP,) and down syndrome critical region 1 (DSCR-1), in conjunction with decreased lung vessel growth may be the initiating factors resulting in pulmonary arterial hypertension, one of the primary pulmonary conditions associated with DS (Fig. 2E) (24). In addition, other antiangiogenic factors whose genes are not located on Hsa21, such as tissue inhibitor of metalloproteinase 3 (TIMP3) and COL4A3 (tumstatin), are upregulated in DS and contribute to impaired vascular development (24). Disrupted vasculature has been shown to contribute to simplified alveolar structures and pulmonary hypoplasia, which is witnessed in a large number of these individuals (Fig. 2C) (20, 24).

Although the increased blood flow often presents as a secondary condition to congenital heart defects and chronic airway obstruction, case studies have shown that is not always true (4). The combination of upper respiratory tract complications in conjunction with pulmonary hypoplasia and compromised capillary system could lead to hypoxia and hypercapnia and ultimately pulmonary hypertension (25). In addition to these manifestations, individuals with DS are at a higher risk of developing pulmonary hemorrhage, a rare lung condition characterized by intra-alveolar bleeding and iron accumulation (Fig. 2F) (26). Interestingly, our recent study demonstrated modulation of the coagulation pathway in prenatal T21 lungs, manifested by downregulation of clotting factors F2 (prothrombin), F5, F7, and F12, as well as vitronectin (VTN), all of which help regulate hemostasis (21). This dysregulation may be contributing to the pulmonary hemorrhage observed in these individuals, as the stability of a platelet plug and clotting become compromised, potentially resulting in bleeding disorders (57). Pulmonary hemosiderosis and isolated pulmonary capillaritis are known to be more severe in individuals with DS (27, 28).

The lymphatic endothelium may also be affected, resulting in lymphangiectasia with dilated pulmonary lymphatics and lymphedema (29). This is established in utero, as dilatation of the lymphatics is first observed in prenatal T21 lungs (21). Although little is known regarding the etiology of this phenomenon, it has been previously shown that there is increased expression of SHH, VEGF-A, and PDGF-B, accompanied by decreased FOXC2 in the jugular lymphatic sacs within DS, which may be indicative of diminished lymphatic differentiation (58). In addition, it should be noted that type I IFN signaling is critical to the maturation of the pulmonary lymphatic endothelium, suggesting its dysregulation in DS may be playing a role in the lymphatic abnormalities (59).

To understand the numerous morphological changes observed in the pulmonary structure of persons with DS, it is first critical to understand how the initial “hits” contribute to these changes. To date, this has been achieved using a combination of in vitro and in vivo models, which, although informative, present with a number of limitations.

MODELS FOR STUDYING DS LUNG DISEASES

There are over 25 mouse models representative of DS, ranging in mutation type (deletion, transchromosomic, translocation, duplication, deletion, etc.) as well as targeted alleles (60), despite the fact that mice lack Hsa21. These models are achieved using the orthologous regions in the mouse to Hsa21, found on mouse chromosomes 10, 16, and 17 (60). However, based on the accumulated evolutional changes in conjunction with altered gene order and orientation from the three orthologous regions on the mouse that have developed into the single Hsa21, no one mouse model truly replicates all of the anomalies associated with DS. Whereas many of these animal models are excellent in demonstrating the neurodegenerative and cardiac phenotypes observed in these individuals, they fail to recapitulate all the hallmarks of human DS lung anomalies and their variability (60–62). Furthermore, it should be noted that numerous differences in lung development have been identified between humans and animal models by our group and others (63–65). Finally, the variability in phenotype penetrance in humans makes the disease very challenging to model in animals.

The use of iPSCs has also proven beneficial in better understanding the complications that individuals with DS face. Induced pluripotent stem cells (iPSCs) offer the unique ability to work with pluripotency and immortality, whereas tissues are limited in both supply and manipulability. These technologies allow to genetically modify or correct trisomy of either single genes or the whole chromosome, thus helping better understand the cellular and molecular mechanisms associated with DS. However, these pluripotent cell types are most often and efficiently differentiated into neural and hematopoietic cells for DS (66, 67). Whereas these are beneficial in understanding the mental complications and leukemogenesis often observed in these individuals, iPSCs continue to prove more complicated when modeling the disease in other cell types. This is due to the variability in efficiency in deriving and differentiating iPSC cell lines. This is an even greater challenge within the DS population, considering the variability within the disease. In addition, iPSCs are limited in studying the interactions between different tissue compartments, which is critical in understanding lung function and development. Thus, a comparison between model systems and native human tissue is required to establish the validity of these model systems to ensure that model system-based conclusions are relevant to human biology. Current cutting-edge technologies such as single-cell RNA sequencing and single-cell proteomics combined with organotypic reconstructions have the potential to provide novel insights into the mechanisms governing developmental lung anomalies in DS.

CONCLUSIONS

Down syndrome is the most common chromosomal anomaly observed, resulting in numerous congenital defects. Although the genotype-phenotype correlation in these individuals has been studied for years, a lot remains to be uncovered. This is partially because, as previously mentioned, not only are the genes expressed on Hsa21 differentially expressed, but their change in expression may alter the expression of other genes as well. We recently demonstrated that a range of lung anomalies is initiated in utero (21). However, to date, the primary areas of research focus in individuals with DS are heart and cognitive/neural development. Considering the large number of persons with DS hospitalized due to pulmonary/respiratory complications and the high morbidity and mortality associated with pulmonary complications, lung development in individuals with DS needs to be better understood, in particular, the contributions of gene modulations associated with T21 to the specific defects observed in utero, as well as the role of novel pathways that have been identified as important in T21 lung development such as the extracellular matrix, coagulation, and interferon pathways.

GRANTS

This work is funded by an American Thoracic Society Unrestricted Pulmonary research grant (to S.D.) and NIH/National Heart, Lung, and Blood Institute Grant R01HL141856 (to D.A.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.D., G.H.D., C.D., T.J.M., and D.A.A. prepared figures; S.D., G.H.D., T.J.M., and D.A.A drafted manuscript; S.D., G.H.D., C.D., T.J.M., and D.A.A. edited and revised manuscript; S.D., G.H.D., C.D., T.J.M., and D.A.A. approved final version of manuscript.