Introduction
In humans, the respiratory tract is divided into the upper airways (nasal cavity, pharynx, and larynx) and the lower airways (trachea, bronchi, bronchioles, and alveoli). The lungs consist of the bronchi, bronchioles, and alveoli. The primary function of the respiratory tract is to efficiently change gas between inhaled air and the bloodstream [
1]. The lungs are constantly exposed to various environmental elements, including pathogens, toxins, and allergens, that cause pulmonary infections or inflammation. Therefore, the human body needs to defend itself against countless intruders through the respiratory tract. Thus, the respiratory system has developed highly sophisticated barrier functions through physiological and immunological mechanisms. Airway epithelial cells (AECs) were originally thought to serve only as a physical barrier. However, recent research has elucidated the interplay between AECs and immune cells, revealing that AECs initiate immune responses [
2]. This complex network of epithelial and immune cells is involved in the pathogenesis of various diseases [
3‐
8]. In fact, inhibitors of thymic stromal lymphopoietin (TSLP), a cytokine mainly produced by AECs in the lungs, have emerged as therapeutic agents for asthma [
9]. Furthermore, there is growing interest in the tripartite network of epithelium, immune, and neural cells [
10‐
14]. In this review, we summarize how the lungs establish sophisticated barrier mechanisms.
Multi-layered lung barriers
The barrier mechanism of the lung is, first, a continuous layer lining the respiratory tract composed of AECs, separating the body from the environment. Second, mucociliary clearance (MCC), consisting of the production of mucus and the coordinated movement of cilia, is the defense mechanism. Third, antimicrobial peptides and proteins act as a chemical barrier. Fourth, various cells, including both immune and non-immune cells, work in a coordinated manner to establish an immunological barrier. By summarizing the function of AECs and innate immune cells as barriers, we will attempt to understand the defense mechanisms in the lung and their involvement in pulmonary diseases.
Airway epithelial cell barriers
AECs play a critical role in establishing the physiological barrier in the lungs. Recent advances in analytical technologies have revealed that AECs are a more heterogeneous population and constitute a more complex network than previously assumed [
41‐
45]. Here, we summarize the characteristics of subpopulations of AECs.
Basal cells
Basal cells exhibit a columnar or cuboidal shape and are firmly anchored to the basement membrane through specialized structures known as hemidesmosomes. They can be identified based on the expression of p63 and keratin 5 (KRT5). Functionally, they are stem cell–like and can self-renew and differentiate into various epithelial cell subtypes, playing a crucial role in maintaining the integrity of the epithelial barrier and facilitating repair and regeneration after injury.
Recent studies have suggested that basal cell abnormalities may contribute to the development of several respiratory diseases. The comparison of RNA-seq data of basal cells between smokers and nonsmokers showed that COPD risk genes identified in GWAS are upregulated by smoking [
46]. Other findings suggest that sensing apoptotic cells by a TAM receptor tyrosine kinase Axl on basal cells is important for tracheal basal cell expansion, cell cycle reentry, and symmetric cell division and is involved in the pathogenesis of COPD [
47]. Meanwhile, single-cell RNA sequencing (scRNA-seq) analysis of lungs from IPF patients identified an aberrant basal cell population that co-expressed basal epithelial, mesenchymal, senescence, and developmental markers [
48]. Another group used single-cell cloning technology to generate a library of basal stem cells from lungs derived from IPF patients [
49]. Among these clones, a clone that transformed normal lung fibroblasts into pathogenic myofibroblasts in vitro was identified. Interestingly, this clone resembled the genetic profile of the abnormal basal cell population identified by scRNA-seq described above [
48,
49]. The relationship between basal cells and allergic pathology has also been the focus of interest. Based on scRNA-seq data of airway epithelial cells derived from patients with chronic rhinosinusitis or asthma, two populations of basal cells with different gene expressions have been reported [
50,
51]. These two populations correspond to differentiation stages, with the less mature population characterized by high expression of TP63. Furthermore, IL-4 and IL-13 have been shown to regulate basal cell stem function in vitro [
50]. In addition, because of their progenitor function, basal cells have attracted attention for their application in regenerative medicine [
52,
53].
Ciliated cells
Ciliated cells are abundant in the large and medium airways and are crucial in moving mucus, which traps debris and pathogens, out of the airways. Each ciliated cell has numerous cilia that extend into the mucus. These cilia are anchored to the cytoskeleton and move in a coordinated manner. They are terminally differentiated and can originate from secretory cells or basal cells. Notch signaling pathways regulate the differentiation of ciliated cells. When Notch signaling is inhibited, it promotes the differentiation of ciliated cells [
54]. Also, forkhead box protein J1 (FOXJ1) is a master regulator of ciliogenesis. Its expression is essential for the differentiation of ciliated cells and the formation of motile cilia. Of clinical importance, ciliated cells have been reported to be involved in viral infections and asthma [
6,
8,
55]. Rhinovirus C (RV-C), the predominant cause of the common cold, is infectious via cadherin-related family member 3 (CDHR3) in the host [
56]. The expression of CDHR3 is shown to be mainly restricted to ciliated cells [
57]. Moreover, CDHR3 has been identified as a susceptibility gene for asthma, especially in young children [
58]. These findings suggest a strong association between rhinovirus infections, CDHR3, and the development of asthma [
55,
58].
Club cells
Club cells, formerly known as Clara cells, are distinctive dome-shaped cubical cells found in the small airways and characterized by the expression of secretoglobin family 1A member 1 (Scgb1a1), also known as Clara cell secretory protein (CCSP). Their differentiation depends on the transforming growth factor-b receptor Alk5 (activin receptor-like kinase 5). They secrete a variety of substances, the most notable being Scgb1a1. This protein has anti-inflammatory and immunosuppressive properties by suppressing various pro-inflammatory cytokines [
59]. In an ALI (air–liquid interface) culture using airway epithelial cells derived from COPD patients, the supplementation of Scgb1a1 was shown to regulate IL-8 release by cigarette smoke extract [
60]. Immunostaining for Scgb1a1 in the airways was reduced in COPD patients and decreased with increasing severity of COPD, suggesting the association between club cells and the pathogenesis of COPD [
61]. It has also been reported that Scgb1a1 in bronchoalveolar lavage (BAL) is decreased in asthma patients [
62]. Of interest, low mRNA expression levels of Scgb1a1 in airway epithelial cell brushings in asthmatic patients have been shown to correlate with poor clinical outcomes [
63]. In addition, club cells possess the ability to differentiate into both ciliated cells and goblet cells. Moreover, in cases where basal cells are injured or lost, club cells can differentiate into basal cells [
64]. Thus, because of the high plasticity of lung epithelial cells, it was difficult to identify the origin cell of the tumor. However, using a lineage-tracking mouse model and scRNA-seq, it has been shown that the causative cells of lung adenocarcinoma are club cells and AT2 cells [
65].
Goblet cells
Goblet cells are characterized by their densely packed mucin granules and surfactant proteins. Their primary function is to produce and secrete mucus. In particular, MUC5AC is mainly synthesized by goblet cells. With ciliated cells, they play a crucial role in facilitating effective MCC. Goblet cells are derived from club cells through the activation of SAM pointed domain containing ETS transcription factor (SPDEF) and forkhead box A3 (FOXA3). In several lung diseases, such as asthma and COPD, there is an increase in the number of goblet cells, a condition known as goblet cell hyperplasia [
66,
67]. This can cause coughing and wheezing due to excessive mucus production. In particular, allergic inflammation and goblet cells are closely related, and IL-13 induces goblet cell hyperplasia and metaplasia [
68,
69].
Pulmonary neuroendocrine cells
Pulmonary neuroendocrine cells (PNECs) can be found either as individual isolated cells or organized in small clusters known as neuroendocrine bodies (NEBs) throughout the conducting airways, especially near the respiratory tree branch. While not being neurons themselves, PNECs are innervated by both the sympathetic and parasympathetic nervous systems. PNECs can monitor airway oxygen and respiratory status and quickly release various substances, including neurotransmitters such as gamma-aminobutyric acid (GABA), calcitonin gene–related peptide (CGRP), bombesin, and serotonin [
70]. These neurotransmitters produced by PNECs have been reported to induce goblet cell hyperplasia and ILC2 activation [
14,
71]. Thus, PNECs are considered to be important in the coordination of the epithelial, immune, and nervous systems, facilitating respiratory homeostasis.
Tuft cells
Tuft cells, also known as brush cells, are expressed in various tissues, including the respiratory and gastrointestinal tracts. Their distinctive morphology, characterized by a tuft of microvilli extending into the mucosal lumen, enables them to sense the extracellular environment. They can detect changes in the local chemical composition and transmit signals to nearby cells [
72,
73]. While the exact function of tuft cells in the lung is still being elucidated, it has been reported that they can release IL-25 and cysteinyl leukotrienes (CysLTs), which synergistically contribute to type 2 inflammation in the lung [
74].
Ionocytes
Recently, pulmonary ionocytes were identified as a rare cell type using scRNA-seq of human bronchial epithelial cells and mouse tracheal epithelial cells [
41,
45]. Ionocytes co-express forkhead box l1 (FOXL1), multiple subunits of the vacuolar-type H
+-ATPase (V-ATPase), and cystic fibrosis transmembrane conductance regulator (CFTR). One of the most notable characteristics is their high expression of CFTR, which encodes an anion channel critical for maintaining the hydration and pH of the airway surface liquid. Thus, they may play an essential role in MCC by cooperating with ciliated cells and secretory cells. Moreover, because mutations in the CFTR gene are responsible for CF, ionocytes have attracted attention as a therapeutic target for CF [
75]. However, it has been recently suggested that the function of CFTR in human airways is mainly carried out by secretory cells, as ionocytes are a small population [
76]. On the other hand, the regulatory function of CFTR-mediated chloride differs between cell types, with secretory cells involved in the secretion of chloride and ionocytes in absorption [
77]. Analysis using the transgenic ferret models suggests that there are at least three subtypes of ionocytes [
78]. Further analysis of ionocytes is expected to clarify the pathogenesis of CF and other airway diseases.
Alveolar cells
The alveolar epithelium consists of two types of epithelial cells: alveolar type1 (AT1) cells and alveolar type 2 (AT2) cells [
16]. AT1 cells are highly specialized for gas exchange and barrier function, characterized by their flat, thin, and squamous morphology. This distinctive shape allows AT1 cells to efficiently cover about 95% of the alveolar surface. AT1 cells, together with capillary endothelium, form the alveolar-capillary barrier, otherwise known as the air-blood barrier. This incredibly thin barrier is crucial not only for efficient gas exchange between the bloodstream carbon dioxide and airborne oxygen but also for separating the bloodstream from foreign pathogens.
AT2 cells are cuboidal cells with apical microvilli and lamellar bodies. Their most important physiological role is the synthesis and secretion of pulmonary surfactant. Pulmonary surfactant reduces surface tension within the alveoli, preventing alveolar collapse during exhalation and preserving alveolar structure for efficient breathing. Besides surfactants, AT2 cells produce various cytokines, chemokines, growth factors, and antimicrobial peptides, which play roles in inflammation, immune responses, and tissue repair.
In addition, AT2 cells serve as progenitor cells for alveolar epithelium. When the alveolar epithelium is injured, AT2 cells can undergo self-renewal and differentiate into AT1 cells, contributing to tissue repair and regeneration [
79]. Wnt signaling may be involved in maintaining the stem cell properties of AT2 cells [
80‐
82]. Furthermore, their regenerative potential is likely to inform the development of novel therapies for COPD [
83] and pulmonary fibrosis [
84,
85].
Two recent reports of scRNA-seq analyses of human distal airways have identified AT0 cells and respiratory airway secretory (RAS) cells. AT0 cells, originated from AT2 cells, can differentiate into AT1 cells or terminal and respiratory bronchiole secretory cells [
43]. In contrast, RAS cells can unidirectional differentiate into AT2 cells through Notch and Wnt signaling [
86]. These cells, which are capable of differentiation, may exhibit phenotypic alterations with age or in response to pathological conditions; thereby, their function and pathology need to be analyzed under a broader range of conditions.
Interactions of epithelial and immune cells in the lung barrier
In recent years, deeper analysis of cellular networks, such as crosstalk between epithelial and immune cells, has become feasible. A contributing factor to this advancement is the progress in transcriptome technologies at the single-cell level, exemplified by scRNA-seq. Furthermore, initially, single-cell transcriptomes had difficulties in data integration and interpretation due to problems such as batch effect, but these problems are being overcome due to large datasets, powerful computational resources, and advances in learning algorithms.
Single-cell transcriptomics offers significant advantages, including the identification of rare cell populations like ionocytes and the estimation of cell differentiation processes through trajectory analysis [
41,
45]. Furthermore, these techniques have enabled the inference of intercellular networks by utilizing information on ligand-receptor interactions [
43,
86]. These advancements are being applied to a variety of respiratory diseases.
Specifically, scRNA-seq conducted on nasopharyngeal and bronchial samples collected from patients with moderate to severe COVID-19 has been reported [
95]. In these patients, secretory cells exhibited significantly higher expression of chemokines such as CXCL1, CXCL3, CXCL6, and CXCL17 compared to controls, suggesting an enhancement in the mobilization of neutrophils, T cells, and mast cells. Furthermore, in severe cases compared to moderate, there was a stronger interaction between epithelial and immune cells, with immune cells, including inflammatory macrophages, being more activated. Thus, this interaction may contribute to the exacerbation of infection. Importantly, infected epithelial cells showed upregulation of the SARS-CoV-2 entry receptor ACE2, which was associated with interferon signaling in immune cells. These findings suggest that intercellular links are important not only in the severity of infection but also in the establishment of infection.
Utilizing a human model of localized asthma exacerbation by bronchoscopic segmental allergen challenge, a comprehensive analysis of the lower airway mucosa of allergic asthmatics and allergic non-asthmatics using scRNA-seq has been conducted [
96]. In response to allergens, asthmatic airway epithelium was highly dynamically upregulating genes involved in matrix degradation, mucus metaplasia, and glycolysis while failing to induce the injury-repair and antioxidant pathways observed in controls. The study revealed a Th2 cell-mononuclear phagocyte-basal cell interactome unique to asthmatics, driven by interactions between TNF family members and type 2 cytokines from Th2 cells. This pathogenic cellular network in asthmatics may override protective injury-repair responses and drive asthma pathobiology.
In addition to the single-cell transcriptome, the advancement of three-dimensional (3D) culture systems, represented by organoids, experimental models that closely recapitulate human physiology, allow further analysis in a coordinated manner [
43,
86,
97]. Fetal lung organoids derived from fetal lung bud tips provide significant insights into lung development. In humans, SOX2 and SOX9 have been identified as progenitor markers, and their maintenance requires EGF, FGF, and WNT signaling, as well as the inhibition of BMP and TGF-β [
98]. The interaction between epithelium and immune cells was also shown to play an important role in lung development using fetal lung organoids [
99]. In immunohistochemistry of fetal lungs, immune cells were found surrounding lung progenitor cells. Therefore, the expression of cytokine receptors in progenitor cells was evaluated, and candidates for interacting cytokines between epithelial and immune cells were identified. Among various cytokines, the supplementation of IL-1β in fetal lung organoids resulted in decreased expression of SOX9 and increased expression of TP63. These findings suggest that myeloid cells, widespread throughout the lungs, produce IL-1β during early lung development, which induces epithelial stem cell differentiation into mature basal cells.
Besides organoids, precision-cut lung slices (PCLS) have emerged in respiratory research as a 3D culture method [
100]. PCLS are live tissue preparations that encompass all resident cell types, including smooth muscle cells, epithelial cells, and fibroblasts. These cells maintain intercellular interactions and cell–matrix relationships within the complex structure of the lung, making them suitable for analysis of cellular networks through single-cell transcriptomics.
Recently, the utilization of human PCLS (hPCLS), single-cell transcriptome analysis, and deep learning-based query-to-reference mapping has been reported as a powerful research platform for elucidating the pathogenesis of lung fibrosis and facilitating drug development [
101]. In this study, fibrosis was induced in hPCLS from nonfibrotic human lung tissue by adding a pro-fibrotic cytokine mix, and the scRNA-seq data obtained from the fibrosis-induced hPCLS were merged with single-cell data from a cohort of pulmonary fibrosis patients. Furthermore, single-cell architectural surgery (scArches), which is a deep learning strategy for mapping single-cell datasets to a reference atlas, was employed to map the obtained data to the Human Lung Cell Atlas, thereby validating an ex vivo model of fibrosis. Additionally, analyses of cell morphology and intercellular networks were conducted through micro-CT staging of hPCLS and patient tissues. The pathways of fibrogenesis and healing processes were evaluated by administering antifibrotic drugs to fibrosis-induced hPCLS. As demonstrated by this cutting-edge research, the analysis of cellular networks in lung physiology and pathogenesis is expected to accelerate with the advancements in transcriptome data analysis, human-like experimental systems, and artificial intelligence technology.
The importance of epithelium as a barrier mechanism in the lungs has been well established. Their central role was thought to be a physical mechanism through cell adhesion and a protective mechanism through mucus production. Indeed, it has been demonstrated that dysfunction of these barrier mechanisms is involved in many pathological conditions, suggesting their importance. Recently, the role of AECs as initiators of immune responses has been recognized. Furthermore, the close relationship between epithelium, neurons, and immune cells is becoming apparent. Further studies are needed to determine the physiological role of this crosstalk and its involvement in the pathogenesis of various lung diseases.
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