REVIEWS Regulation of immune responses by the airway epithelial cell landscape Richard J. Hewitt and Clare M. Lloyd ✉ Abstract | The community of cells lining our airways plays a collaborative role in the preservation of immune homeostasis in the lung and provides protection from the pathogens and pollutants in the air we breathe. In addition to its structural attributes that provide effective mucociliary clearance of the lower airspace, the airway epithelium is an immunologically active barrier surface that senses changes in the airway environment and interacts with resident and recruited immune cells. Single-cell RNA-sequencing is illuminating the cellular heterogeneity that exists in the airway wall and has identified novel cell populations with unique molecular signatures, trajectories of differentiation and diverse functions in health and disease. In this Review, we discuss how our view of the airway epithelial landscape has evolved with the advent of transcriptomic approaches to cellular phenotyping, with a focus on epithelial interactions with the local neuronal and immune systems. It is now well accepted that the cells lining the airways composition reflect this. Specialized epithelial cell popu­ constitute more than just a barrier between the exter­ lations line the entire respiratory tract from the nasal nal environment and the underlying mesenchyme. cavity to the alveoli. Elegant electron microscopy studies This collection of specialized epithelial cells responds provided early insight into the morphology and ultra­ to microbes and noxious stimuli that overcome the structure of the principle epithelial cell types residing in mucociliary barrier and are a vital component of host the human airways2–4. In addition to using classical elec­ defence, interacting with cells of the immune system to tron microscopy­defined morphological features, stand­ maintain homeostasis while facilitating immune reac­ ard immunohistochemical staining for cell type­specific tions when necessary1. The respiratory epithelium must markers has been used to characterize and quantify epi­ also manage responses to the diverse toxins contained thelial cell populations throughout the human respiratory within the inhaled environment and there is emerg­ tract, determining the influence of anatomical location on ing evidence that epithelial dysfunction is a driver of cellular composition5–7. The airways are lined by ciliated numerous chronic diseases affecting the lungs. The tra­ and secretory cells primarily adapted to facilitate muco­ ditional view of the epithelial layer incorporates basal ciliary clearance of particulate matter and infectious path­ cells in close proximity to secretory and ciliated cells, ogens in the air we breathe. Like other mucosal surfaces, forming a tight unit that maintains a physical barrier the airway epithelium is at an interface with the environ­ but is also responsive to the inhaled environment via ment and is therefore critically important to host defence. cells and molecules from the immune system. However, Despite sharing the same embryological origin as the gut with the advent of advanced sequencing techniques, this mucosa, immunological activity at the airway mucosal view has changed to one of a dynamic cellular structure surface is necessarily distinct and shaped by differences encompassing a wide range of highly specialized cells in environmental conditions (temperature gradient, bidi­ that are able to respond to environmental change, inter­ rectional airflow), resident microbial communities and act with resident microbial communities and cooperate airborne antigens8. Although not the focus of this Review, with multiple other specialized cellular systems such as the epithelial cells lining the distal alveolar region of the the immune and neural systems. lung are phenotypically and functionally distinct; alveolar The respiratory tract is a complex organ system epithelial type 1 (AT1) cells provide a specialized surface National Heart and Lung divided into the upper respiratory tract, that includes the for gas exchange and type 2 (AT2) cells secrete pulmonary Institute, Imperial College London, London, UK. nasal cavity, pharynx and larynx, and the lower respira­ surfactant to prevent alveolar collapse during expiration. ✉e-mail: c.lloyd@ tory tract comprising the conducting airways (trachea, In each anatomical niche, there are progenitor cell popula­ imperial.ac.uk bronchi and bronchioles) and the respiratory zone (res­ tions, for example, basal cells in the airways and AT2 cells https://doi.org/10.1038/ piratory bronchioles and alveoli) (Fig. 1). Each area has a in the alveoli, that ensure robust epithelial regeneration s41577-020-00477-9 specific function and the regional differences in cellular under homeostatic conditions and following injury9. NATURE REVIEWS | IMMUNOLOGY VOLUME 21 | JUNE 2021 | 347 REVIEWS Traditional landscape Contemporary landscape Club Ciliated Goblet Deuterosomal Club Ciliated Tuf Goblet cell cell cell cell cell cell cell cell Pulmonary Basal neuroendocrine Basal Suprabasal Ionocyte cell cell cell cell Anatomical region Method of obtaining samples for scRNA-seq studies Nasal cavity Upper Airway respiratory Pharynx tract Nasal brushings Larynx and biopsies Trachea Brushings and endobronchial biopsies via bronchoscopy Lower respiratory BAL via bronchoscopy tract Bronchi Bronchioles Parenchymal tissue Surgical lung biopsy Respiratory zone Alveoli and explants Lungs Fig. 1 | Revised cell repertoire of human airway epithelium captured by and the presence of disease. The approach used to obtain human samples scRNA-seq. The traditional view of the airway epithelium — comprising for sequencing differs according to lung compartment; the principle epithelial cells lining the upper and lower respiratory tract — has been methods for sampling human airway epithelial cells are bronchial brushings transformed by single-cell RNA sequencing (scRNA-seq). The revised con- and endobronchial biopsies conducted during bronchoscopy, in contrast to temporary landscape features newly identified cell types, such as the iono- sampling of the alveolar region, which is achieved using parenchymal lung cyte, and different cell states. The cellular composition and cell states within tissue obtained from surgical biopsy or from explants. BAL, bronchoalveolar the airway varies according to anatomical location (proximal–distal axis) lavage. The advent of novel sequencing techniques has cell subsets in a heterogenous cell population, driving facilitated not only the identification of novel cell types the discovery of unidentified cell types and states11–13. The but reveals potential functions of previously named but Human Cell Atlas Consortium aims to comprehensively poorly understood cell types, for example, tuft/brush chart — at single­cell resolution with integration of spa­ Bronchoscopy cells. There is also an indication that heterogenous cell tial data — the changes that occur in lung cell compo­ A procedure in which a types and states exist and that subtle changes in these sition and molecular phenotype in health and disease14. fibre-optic camera is used will be influenced by the changing environment, for Added to this, it is also possible to computationally to visualize the airways. example, by smoking or exposure to allergens or pol­ interrogate epithelial–immune interactions in the airway 10,15–17 Transcriptome lutants. It is now clear that the airway wall represents a niche using known ligand–receptor interactions . All the RNA transcripts dynamic ‘community’ of epithelial cells existing in close Studying human epithelial biology using single­cell expressed in a cell or association with resident immune and neuronal cells to transcriptomics relies on the availability of lung tissue. population of cells. generate an integrated unit that plays a critical role in One of the earliest studies in humans analysed diseased Human Cell Atlas maintaining mucosal immune homeostasis as well as explant parenchymal lung tissue from patients with Consortium facilitating host defence against inhaled pathogens. In idiopathic pulmonary fibrosis (IPF) and lung lobes from An international community of this Review, we outline how single­cell transcriptomic donors unsuitable for transplant as control tissue18. For scientists collaborating with the techniques have been used to map the airway epithelial studies of the human airway epithelium, the acquisition primary goal of characterizing bronchial brushings endobronchial biopsy all human cell types by landscape and how this has highlighted the function of and samples unique gene expression profile, of novel specialized epithelial cells and how they inter­ in the setting of disease can be achieved through fibre­ developmental trajectories act with cells of the immune and neuronal systems to optic bronchoscopies carried out in the context of routine and spatial localization. regulate airway immunity. clinical care10,19. It is important to note that the cellular Idiopathic pulmonary content differs by the sampling method used and this fibrosis Mapping the airway epithelial landscape will influence the cell clusters obtained by scRNA­seq (IPF). Progressive, scarring lung Single­cell RNA­sequencing (scRNA­seq) has trans­ (Fig. 2). Acquiring age­matched, healthy control samples disease driven by alveolar formed our view of the airway epithelium, unveiling a is more challenging as it necessitates research volunteers epithelial cell injury, fibroblast level of cellular diversity that had