Respiratory tract

The respiratory system is divided into the upper and lower respiratory tract. The nasal cavity, pharynx, and larynx are part of the upper airway tract, whereas the trachea, bronchial tree, and lungs belong to the lower airways. The trachea divides into two primary bronchi, each leading into a lung, where they branch into smaller bronchi. As bronchi become narrower, they are considered as bronchioles, which terminate into alveoli that are responsible for gas exchange. The respiratory tract is lined with an epithelium that changes its cellular composition along the proximal to distal axis (Figure 2a).

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Figure 2: Cellular composition of the murine airway epithelium.

a) The cellular composition of the airway epithelium differs along the proximal-distal axis. The epithelium of the conducting airways mainly consists of basal, club, and ciliated cells. Rare cell types are goblet, brush, ionocyte, and neuroendocrine cells. Notice that further down the bronchial tree, basal cells are absent in the murine lung. b) Airway epithelial cells can regenerate the respiratory epithelium under homeostatic conditions. Basal cells have the capacity of self-renewal and differentiate into all different kinds of airway epithelial cells. Hence, basal cells are the stem cells of the large airways. Progenitor cells of the small airways are club cells, which can transdifferentiate into ciliated and goblet cells. Adapted from (Rackley and Stripp, 2012; Schilders et al., 2016; Montoro et al., 2018).

The trachea and bronchi are covered by a pseudostratified columnar epithelium composed of a variety of cell types, which mainly include basal, ciliated, and non-ciliated secretory cells (club cells). In contrast, smaller airways are predominantly comprised of ciliated and club cells that form the columnar epithelium (Rackley and Stripp, 2012). Moreover the conducting airways are composed of goblet, brush, ionocyte, hillock (not shown in Figure 2), and neuroendocrine cells, which are quite rare cell type populations (Montoro et al., 2018). Additionally, the alveolar epithelium contains alveolar type I and II cells (pneumocytes).

In general, these various cell types are found in human and mouse airways; however, the distribution differs slightly between these two species. Basal cells are restricted to the

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tracheal region in mice, whereas in humans, they are found throughout the epithelium with their amount declining with airway size (Rackley and Stripp, 2012).

Arrangement of the individual lung lobes is another pivotal difference between the human and mouse respiratory tract. The human lung is segmented into three lobes on the right und two lobes on the left, while the murine lung consists of four right lobes and a single left lobe (Fox et al., 2006).

All cells of the respiratory epithelium function together to protect against the entry of foreign pathogens or particles by not only acting as a physical barrier and mucociliary escalator but also by contributing to the innate immune response. The innate immune response of the respiratory epithelium is achieved by the secretion of soluble factors such as cytokines and chemokines by airway epithelial cells. These inflammatory mediators recruit immune cells and thereby activating the innate and adaptive immunity. Thus, the airway epithelium contributes directly to the host defense. Due to the constant exposure of airways to external stimuli, it is important that the airway epithelium is renewed upon injury to restore its functions (Tam et al., 2011).

1.2.1.1 Respiratory epithelial cells and their function

The various respiratory epithelial cells including basal, club, multiciliated, goblet, neuroendocrine, brush, ionocyte, and hillock cells are vital for maintaining airway homeostasis and regeneration (Rackley and Stripp, 2012; Montoro et al., 2018; Plasschaert et al., 2018).

Basal cells

Basal cells are a population of undifferentiated progenitor cells that cover the basement membrane without being exposed to the airway lumen. These progenitor cells are considered to be the stem cells of the airways due to their ability to differentiate into all the distinct cell types that form the respiratory epithelium (Figure 2b). Hence, basal cells contribute to the homeostasis and repair of the large airways of the epithelium due to their predominant expression there. In contrast, in the lower respiratory tract, club cells are the main progenitor cells (Rackley and Stripp, 2012).

Club cells

Club cells are predominantly found in the small airways. They can proliferate and differentiate into ciliated and goblet cells (Figure 2b). Apart from their role as progenitor cells, club cells secrete proteins such as the club cell 10 kDa protein (CC10) (Hiemstra and Bourdin, 2014) and surfactant proteins (SP-A, SP-B, and SP-D) into the fluid lining the respiratory bronchioles (Han and Mallampalli, 2015). Surfactant proteins are essential for lowering the surface tension at the alveolar and bronchiolar air-liquid interface (ALI) (SP-B,

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SP-C), but also contribute to the host defense (SP-A, SP-D) (Han and Mallampalli, 2015).

Furthermore, club cells are the only cell type of the respiratory epithelium that express P450 monooxygenases necessary for the detoxification of substances, including the polycystic aromatic hydrocarbon naphthalene (described in section 1.5.1.1) (Tam et al., 2011).

Multiciliated cells

MCCs are situated in the epithelium of the small and large airways and arise from club cells and basal cells respectively (Tam et al., 2011; Montoro et al., 2018). Under homeostatic condition, MCCs are differentiated columnar cells. However, ciliated cells can transdifferentiate into goblet cells during inflammatory processes as ciliated/goblet cell transdifferentiation is mediated by inflammatory cytokines (Tyner et al., 2006; Gomperts et al., 2007; Turner et al., 2011).

The coordinated beating of multiple motile cilia on MCCs contributes to the mucociliary clearance by transporting pathogens or particles trapped in the mucus out of the airways (Tam et al., 2011). Consequently, defective motile cilia trigger the onset of airway diseases such as primary ciliary dyskinesia (PCD) and reduced generation of multiple motile cilia, which is another mucociliary clearance disorder (Boon et al., 2014; Wallmeier et al., 2014).

Moreover, dysfunctions of motile cilia are also involved in the pathogenesis of acquired airway diseases such as chronic rhinosinusitis, chronic bronchitis, and chronic obstructive pulmonary disease (COPD) (Tilley et al., 2015) (described in section 1.4).

Efficient mucociliary clearance depends not only on the movement of motile cilia but also on the amount and viscoelasticity of the periciliary layer and the overlying mucus. The periciliary layer surrounds motile cilia, supports coordinated cilia movement, and serves as a barrier between the mucus trapped particles and the cell surface. Impairment of the periciliary layer (e.g. by dehydration) results in the collapse of the mucus layer, which in turn leads to defective mucociliary clearance as observed in cystic fibrosis (described in section 1.2.1.1). The mucus layer is mainly composed of glycoproteins, which are secreted by goblet cells and submucosal glands (Bustamante-Marin and Ostrowski, 2017).

Goblet cells

Goblet cells are present only in small numbers in the conducting airways and help in regulating mucus production. In response to inflammatory signals (e.g. cytokines) the number of goblet cells increase (metaplasia, hyperplasia), which results in hypersecretion of mucus that in turn triggers airway obstruction associated with chronic airway diseases such as chronic bronchitis (Rackley and Stripp, 2012).

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Neuroendocrine cells

Pulmonary neuroendocrine cells are present throughout the respiratory epithelium and are commonly found at innervated airway branch points, where they often form clusters, which are termed neuroepithelial bodies (Rackley and Stripp, 2012). Neuroendocrine cells possess neuro-immunomodulatory functions and are required for allergen-induced responses (Sui et al., 2018). Increased numbers of neuroendocrine cells have been found in diseases associated with chronic inflammation such as COPD (Gu et al., 2014).

Moreover, the microenvironment of the neuroepithelial body functions in maintaining progenitor cells capable of epithelial regeneration after injury for example by naphthalene (described in section 1.5.1.1) (Reynolds et al., 2000).

Brush cells

Brush cells (also called tuft cells) are characterized by the presence of microvilli (Reid et al., 2005). They have been less intensively studied. Until now, it has been shown that brush cells line the entire airway and alveolar epithelium (Reid et al., 2005) and act as epithelial chemosensors by detecting irritants via the canonical taste transduction cascade (Krasteva et al., 2011).

Ionocyte and hillock cells

Recently, two new cell types of the respiratory epithelium have been identified, named ionocytes and hillocks (Montoro et al., 2018; Plasschaert et al., 2018). Ionocytes represent a rare population of pulmonary cells that function in fluid regulation at the airway surface due to their high expression of the chloride channel called cystic fibrosis transmembrane conductance regulator (CFTR) (Montoro et al., 2018; Plasschaert et al., 2018). Mutation in CFTR gene are associated with the pathophysiology of cystic fibrosis. Cystic fibrosis is a multiorgan disease, which is characterized amongst other things by chronic airway infections. Dysfunction of CFTR leads to several changes in the airways, including dehydration and acidification (Boucher, 2007; Shah et al., 2016). Dehydration of the airway surface fluid caused by an increased fluid absorption into the cell is accompanied by a series of consecutive events, including collapse of the periciliary fluid layer, increased mucus viscosity, and impaired mucociliary clearance (Boucher, 2007).

The second newly identified cell type is the hillock cell, which is characterized by a high cellular turnover that is linked to immunomodulation and barrier function (Montoro et al., 2018).

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