key: cord-0753787-txvlmzrn
authors: Hewitt, Richard J.; Lloyd, Clare M.
title: Regulation of immune responses by the airway epithelial cell landscape
date: 2021-01-13
journal: Nat Rev Immunol
DOI: 10.1038/s41577-020-00477-9
sha: 8cdbb52e8b1c021a9fc49c31d9c62196dfb19f5f
doc_id: 753787
cord_uid: txvlmzrn

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 constitute more than just a barrier between the exter nal environment and the underlying mesenchyme. This collection of specialized epithelial cells responds to microbes and noxious stimuli that overcome the mucociliary barrier and are a vital component of host defence, interacting with cells of the immune system to maintain homeostasis while facilitating immune reac tions when necessary 1 . The respiratory epithelium must also manage responses to the diverse toxins contained within the inhaled environment and there is emerg ing evidence that epithelial dysfunction is a driver of numerous chronic diseases affecting the lungs. The tra ditional view of the epithelial layer incorporates basal cells in close proximity to secretory and ciliated cells, forming a tight unit that maintains a physical barrier but is also responsive to the inhaled environment via cells and molecules from the immune system. However, with the advent of advanced sequencing techniques, this view has changed to one of a dynamic cellular structure encompassing a wide range of highly specialized cells that are able to respond to environmental change, inter act with resident microbial communities and cooperate with multiple other specialized cellular systems such as the immune and neural systems.

The respiratory tract is a complex organ system divided into the upper respiratory tract, that includes the nasal cavity, pharynx and larynx, and the lower respira tory tract comprising the conducting airways (trachea, bronchi and bronchioles) and the respiratory zone (res piratory bronchioles and alveoli) (Fig. 1) . Each area has a specific function and the regional differences in cellular composition reflect this. Specialized epithelial cell popu lations line the entire respiratory tract from the nasal cavity to the alveoli. Elegant electron microscopy studies provided early insight into the morphology and ultra structure of the principle epithelial cell types residing in the human airways [2] [3] [4] . In addition to using classical elec tron microscopydefined morphological features, stand ard immunohistochemical staining for cell typespecific markers has been used to characterize and quantify epi thelial cell populations throughout the human respiratory tract, determining the influence of anatomical location on cellular composition [5] [6] [7] . The airways are lined by ciliated and secretory cells primarily adapted to facilitate muco ciliary clearance of particulate matter and infectious path ogens in the air we breathe. Like other mucosal surfaces, the airway epithelium is at an interface with the environ ment and is therefore critically important to host defence. Despite sharing the same embryological origin as the gut mucosa, immunological activity at the airway mucosal surface is necessarily distinct and shaped by differences in environmental conditions (temperature gradient, bidi rectional airflow), resident microbial communities and airborne antigens 8 . Although not the focus of this Review, the epithelial cells lining the distal alveolar region of the lung are phenotypically and functionally distinct; alveolar epithelial type 1 (AT1) cells provide a specialized surface for gas exchange and type 2 (AT2) cells secrete pulmonary surfactant to prevent alveolar collapse during expiration. In each anatomical niche, there are progenitor cell popula tions, for example, basal cells in the airways and AT2 cells in the alveoli, that ensure robust epithelial regeneration under homeostatic conditions and following injury 9 .

The advent of novel sequencing techniques has facilitated not only the identification of novel cell types but reveals potential functions of previously named but poorly understood cell types, for example, tuft/brush cells. There is also an indication that heterogenous cell types and states exist and that subtle changes in these will be influenced by the changing environment, for example, by smoking or exposure to allergens or pol lutants. It is now clear that the airway wall represents a dynamic 'community' of epithelial cells existing in close association with resident immune and neuronal cells to generate an integrated unit that plays a critical role in maintaining mucosal immune homeostasis as well as facilitating host defence against inhaled pathogens. In this Review, we outline how singlecell transcriptomic techniques have been used to map the airway epithelial landscape and how this has highlighted the function of novel specialized epithelial cells and how they inter act with cells of the immune and neuronal systems to regulate airway immunity.

Mapping the airway epithelial landscape Singlecell RNAsequencing (scRNAseq) has trans formed our view of the airway epithelium, unveiling a level of cellular diversity that had not been documented using microscopy for phenotyping 10 . This technology profiles the transcriptome of individual cells and there fore facilitates an unbiased characterization of different cell subsets in a heterogenous cell population, driving the discovery of unidentified cell types and states [11] [12] [13] . The Human Cell Atlas Consortium aims to comprehensively chart -at singlecell resolution with integration of spa tial data -the changes that occur in lung cell compo sition and molecular phenotype in health and disease 14 . Added to this, it is also possible to computationally interrogate epithelial-immune interactions in the airway niche using known ligand-receptor interactions 10, [15] [16] [17] .

Studying human epithelial biology using singlecell transcriptomics relies on the availability of lung tissue. One of the earliest studies in humans analysed diseased explant parenchymal lung tissue from patients with idiopathic pulmonary fibrosis (IPF) and lung lobes from donors unsuitable for transplant as control tissue 18 . For studies of the human airway epithelium, the acquisition of bronchial brushings and endobronchial biopsy samples in the setting of disease can be achieved through fibre optic bronchoscopies carried out in the context of routine clinical care 10, 19 . It is important to note that the cellular content differs by the sampling method used and this will influence the cell clusters obtained by scRNAseq (Fig. 2 ). Acquiring agematched, healthy control samples is more challenging as it necessitates research volunteers undergoing an invasive procedure. The procurement of appropriate human lung tissue is only feasible through close collaboration between clinicians and scientists and through human tissue biobanks. Importantly, logistical and the presence of disease. The approach used to obtain human samples for sequencing differs according to lung compartment; the principle methods for sampling human airway epithelial cells are bronchial brushings and endobronchial biopsies conducted during bronchoscopy, in contrast to sampling of the alveolar region, which is achieved using parenchymal lung tissue obtained from surgical biopsy or from explants. BAL, bronchoalveolar lavage.

A procedure in which a fibre-optic camera is used to visualize the airways.

All the RNA transcripts expressed in a cell or population of cells.

An international community of scientists collaborating with the primary goal of characterizing all human cell types by unique gene expression profile, developmental trajectories and spatial localization.

(iPF). Progressive, scarring lung disease driven by alveolar epithelial cell injury, fibroblast activation and excessive extracellular matrix deposition in the interstitium, impairing gas exchange.

www.nature.com/nri practicalities require careful consideration because the hypothermic preservation of tissue and the minimiza tion of time delays between fresh sample collection in the clinic and processing in the laboratory are critical to ensuring highquality scRNAseq data. A cold ischaemic time of 72 h results in an increase in the presence of mito chondrial reads due to cellular stress or death in multiple lung cell types and a cellspecific decrease in the propor tion of CD4 + and CD8 + cytotoxic T cells 20 . The enzymatic dissociation of tissue with collagenase is widely used to isolate cells for scRNAseq studies but, as a caveat, it can induce the expression of immediate early genes, includ ing Fos and Jun, and heat shock protein (HSP) genes in a subpopulation of cells, thus also potentially introducing an important artefact and potential bias in singlecell data sets 21 . During scRNAseq analysis, cells sharing similar gene expression profiles are clustered together and can be assigned an annotation based on known marker genes 11, 13 (TAble 1 ). In the first singlecell studies of the mammalian airway, a new cluster of cells was identified that did not map to any of the previously known epithe lial cell types and, based on its gene expression signature, was termed the 'pulmonary ionocyte' 22, 23 . This sentinel discovery of a novel cell type highlights the power of scRNAseq (boxes 1,2).

Subclustering can be used to identify diverse cell states within a specific celltype cluster. A singlecell analy sis of human nasal epithelial cells differentiated in air-liquid interface cultures demonstrated a precursor sub set of FOXJ1expressing multiciliated cells termed 'deu tro somal cells' , which express specific genes, including DEUP1 (also known as CCDC67), FOXN4 and CDC20B, all of which are critical for centriole amplification and ciliogenesis 24, 25 . Deutrosomal cells are identifiable in the healthy human airways by scRNAseq 19 .

Subtle but distinct molecular states of basal, goblet and ciliated cells denoted by differential marker gene expression have now been described in the human airway 10 . Goblet cells could be divided into two subsets according to their uniquely expressed genes; for exam ple, 'goblet 2' cells in the nasal epithelium have higher expression of genes involved in immune cell recruitment such as CXCL10, IL19 and CSF3 (ReF. 10 ). The anatomical origin of the cell population within the respiratory tract plays an important role in governing its transcriptional signature and cell state. A singlecell study of brush ings and biopsies taken from four distinct anatomical For human single-cell RNA sequencing (scRNA-seq) studies of the lower respiratory tract, a flexible bronchoscopy is performed to obtain bronchoalveolar lavage (BAL) fluid, airway wall brushings and endobronchial biopsies. The starting material determines the relative contributions of airway immune cells, epithelial cells and underlying stromal cells that are sequenced. A viable, single-cell suspension is obtained by enzymatic dissociation of brushings and biopsies. Bioinformatic pipelines generate distinct cell clusters by dimensional reduction and visualization techniques such as uniform manifold approximation and projection (UMAP) and facilitate assignment of cluster annotations based on cell-marker genes. Novel cell clusters and cell states can be determined in health and disease and trajectory analysis can be used to study dynamic, differentiating cell types.

A procedure in which a brush is used through a bronchoscope to obtain cells lining the airway under direct visualization.

A procedure in which a small biopsy is performed using forceps through a bronchoscope to obtain a sample of airway wall cells under direct visualization.

Air-liquid interface cultures epithelial cells are cultured on a microporous membrane in a Transwell plate and exposed to air to allow mucociliary differentiation.

locations in healthy subjects, highlighted regionspecific subclusters of suprabasal, secretory and ciliated cells between nasal and tracheobronchial compartments 19 . Spatial variations in gene expression profiles between cells of the same type relate, in part, to the biological function required at a specific airway location. Secretory cells in the nasal epithelium are enriched in gene sets associated with cell motility, differentiation and sensory perception pathways, whereas in the bronchial tree, they are enriched in genes related to innate immunity and wound healing response pathways 19 . Noteworthy is the inconsistent annotation of secretory cell types, including club cells and goblet cells, between singlecell studies due to overlapping gene expression signatures 10, 19, 26 .

A further determinant of airway epithelial cell state is revealed by the comparison of cells from healthy volun teers versus those collected from patients with respiratory disease. An analysis of airway wall biopsies from mildto moderate asthmatics exposed several cell states not found in the healthy airway 10 . Cells coexpressing gene markers of ciliated cells, including FOXJ1, and goblet cell genes such as MUC5AC, were termed 'mucous ciliated cells' and are postulated to represent a novel transitionary cell state driven by IL4/IL13 signalling in asthma.

Singlecell analyses of parenchymal lung tissue from patients with IPF, a progressive scarring lung disease, have reported a previously unidentified, transcription ally distinct cluster of cells in the fibrotic distal lung that express some but not all markers of airway basal cells 27, 28 . These intriguing 'aberrant basaloid' cells, which are KRT5 -KRT17 + , coexpress genes encoding mesenchymal markers such as collagen type 1 α1 chain (COL1A1), the transcription factor SOX9 (which plays a role in distal air way development) and genes linked to IPF pathogenesis, including MMP7, which encodes matrilysin.

The pulmonary epithelium represents a dynamic cellular community and singlecell transcriptomics cap tures a unique snapshot of cells at different stages in the continuous process of differentiation -a feature that was first harnessed to elucidate epithelial lineage hier archies at four distinct stages of alveolar development in mice 29 . With the refinement of scRNAseq bioinfor matic approaches it has been possible to study cell fate in human samples and to unveil the subtleties of human airway epithelial cell differentiation 10, 24, 30 . The transcrip tional changes that accompany the differentiation of each cell can be exploited computationally by trajectory inference algorithms that order cells according to pro gress along their individual trajectories, which is quanti fied by 'pseudotime' 31, 32 . A pseudotime analysis of nasal airway epithelial cells in air-liquid interface cultures demonstrated that goblet cells could act as precursors to ciliated cells and that transitory FOXJ1 + MUC5AC + cells could be found in the context of this differentia tion trajectory 24 . Furthermore, leveraging the relative abundance of unspliced and spliced mRNA per gene, it is possible to estimate the rate of gene expression change, termed 'RNA velocity' , and to predict future cell states 33, 34 .

Pseudotemporal ordering of cells using a diffusion map approach 35 identified an intriguing population of Krt13expressing cells in the mouse airway as an intermediary in basal to club cell differentiation 23 . This transitional cell was arranged in discrete, stratified, highturnover structures termed 'hillocks' and expressed www.nature.com/nri genes associated with squamous epithelial differentiation, cell adhesion and immunomodulation. The comprehensive transcriptomic information charted at a cellular level by scRNAseq has heightened our knowledge of the airway epithelial landscape at steady state. This technology has also been harnessed to study airway samples from critically ill patients with COVID19 in order to provide finer grain insight into the pathogenesis of severe acute respiratory system coronavirus 2 (SARSCoV2) infection 16, 36, 37 . Studying singlecell gene expression in multiple tissue types from data sets collated from the Human Cell Atlas Consortium, genes associated with SARSCoV2 cell entry, namely ACE2, which encodes a viral entry recep tor, and TMPRSS2, were significantly enriched in nasal secretory and ciliated epithelial cells. Noteworthy was the high ACE2 expression in cells at other mucosal sur faces, including the cornea, conjunctiva, small intes tine and colon, which may, in combination with high nasal expression, explain the higher transmissibility of SARSCoV2 (ReF. 36 ). This demonstrates how existing data sets can be exploited to further our understanding of the pathogenesis of a novel emerging disease.

Functions of rare epithelial cell types Basal cells are multipotent stem cells that account for a third of all airway epithelial cells and give rise to other major subpopulations, such as secretory and ciliated cells, during homeostatic maintenance of the epithelial barrier and regeneration in response to injury 38 . Basal cells are anchored to the basement membrane of the conducting airways and express keratin 5 (KRT5) and transcription factor tumour protein p63 (TP63). Lineage tracing and singlecell transcriptomic stud ies have demonstrated that functionally distinct basal cell subsets exist in the airway 24, 39 . Two discrete basal cell states are present in the upper and lower airways, but their frequency is lower in the upper airways. These cell states reflect distinct differentiation stages in basal cells, with mature basal cells (located at a more apical region) expressing lower levels of TP63 and NPPC than the less mature basal cells 10 . Basal cells with a lower TP63 expres sion and higher KRT19 and NOTCH3 expression were annotated as 'suprabasal' cells by Deprez et al. 19 .

In addition to these major cell types, rarer cell popu lations comprising less than 1% of all human airway epi thelial cells have been identified via detailed singlecell transcriptomic analyses 19, 26 . Although uncommon, these cells seem to have very distinct phenotypes and perform specific tasks that are vital for the efficient functioning and maintenance of the epithelial landscape.

The advent of scRNAseq has enabled investigators to examine cell development trajectories. In addition to the connection between basal, club and ciliated cells, Montoro et al. defined a distinct trajec tory, which links tracheal basal and club cells via a novel transitional cell that uniquely expresses Krt13 and Krt4 (ReF. 23 ). By looking at the location of these cells within the lungs coupled with lineage tracing in mice, the authors noted that Krt13 + cells lie within contiguous groups of stratified cells that do not contain luminal ciliated cells. They termed these groupings 'hillocks' , a structure composed of luminal Scgb1a1 + Krt13 + cells lying on top of Trp63 + Krt13 + basal cells, with the expres sion of Trp63 occurring along a gradient from basal to suprabasal divisions. Critically, hillocks contain a par ticularly high number of cycling cells and expressed markers of cellular adhesion and epithelial differentia tion as well as genes associated with barrier function and immunomodulation 23 . A parallel study by Plasschaert et al. confirmed the presence of a distinct population of Krt13 + Krt4 + cells in the murine trachea and in ex vivo differentiated human basal epithelial cells, although they concluded that this population represented an intermediate population between basal stem cells and differentiated luminal secretory cells 22 . Furthermore, Krt13 + Krt4 + expressing cells emerged in the tracheal epithelium 1 day after polidocanolinduced injury in mice 22 . In humans, KRT13 is expressed in the airway epithelium by a subpopulation of 'cycling' basal cells that are characterized by the expression of genes asso ciated with cell proliferation, including MKI67 (which encodes Ki67) 10 . Further analyses are needed to confirm whether hillocks represent a truly distinct niche rather than a metaplastic zone in transition and to determine their origin and purpose. Indeed, it is not clear whether these structures are present throughout the airways or are restricted to the trachea. In support of their exist ence, Deprez et al. describe a population of KRT13 + cells, a high proportion of which were cycling, in nasal Box 1 | unanswered questions the field of single-cell transcriptomics has provided a transformative, high-definition view of the cellular communities that exist in the human airway but has also generated many unanswered questions for the research community.

• How do cells in the contemporary single-cell landscape reflect those detected by traditional microscopy methods? Data from single-cell rNa sequencing has refined our characterization of historically described airway cell types, such as tuft cells 4 , and has led to the discovery of a previously undescribed cell -the 'ionocyte' 22, 23 . electron microscopy studies in 1994 reported 'cells of indeterminate type' 5 and there remain undefined cell clusters in recent single-cell data sets 16, 19 ; therefore, future work must focus on better characterizing these cell types as well as on determining their spatial relationships within the airway wall during health and disease. • How much significance should we place on the discovery of clusters with low cell abundance or sub-clusters of a specific cell type? in addition to confirming the existence of a specific cell type through immunofluorescence microscopy, to truly bring gravity and biological relevance, a functional difference should be experimentally demonstrated 23, 26 . the challenge is then to interpret these findings in the context of human health and disease. • How do we account for the inherent biological variability between human samples and cell clusters that arise in a minority of study subjects? One approach to provide greater transparency is to show cellular composition and clusters for individual subjects as well as those integrated by disease 19, 139 . • How do we reconcile differences in the assignment of cell identification? Consensus must be reached on specific positive and negative marker genes that define a particular cell type because there are discrepancies in the annotation of cell types and subsets 10, 16, 19, 26 . Consistency and clarity on the anatomical origin of the airway cells should also be factored into any new classification system. • Cell fate mapping has traditionally relied on the use of reporter mice, but now various computational tools allow the inference of cell trajectory from human single-cell data 32 . Differences have been reported in lineage relationships between basal cells and rare cell types (tuft cells, pulmonary neuroendocrine cells and ionocytes), which may reflect the species studied (mouse versus human), model and/or trajectory algorithm used 23,26 but also highlights the need for further validation in human samples.

Nature reviews | Immunology turbinates, indicating that hillock cells may exist in other areas of the human respiratory tract 19 .

The respiratory tract contains several groups of chemosensory epithelial cells that coordinate interactions with the external environment -the enteroendocrine cells and the tuft cells (also known as 'brush' cells). These chemosensory epithelial cells share similarities with taste cells and are predicted to evoke both positive and negative responses from immune and neuronal cells 40 .

They have a distinctive morphology, being bottle shaped with apical microvilli, and are expressed in a range of organs, including the gut and airways. Their existence is well documented in the gut, where they are thought to be triggered by dietary metabolites such as succinate and bacterial products (for example, quorumsensing lactones or damino acids) [41] [42] [43] . By contrast, their role in the lung is less certain, but they have been detected in the nose, trachea and proximal airways and exist in close contact with nerve fibres, mediating communica tion between neuronal and immune pathways. However, they are not universally identifiable; indeed, Vieira Braga et al. 10 could not identify a unique population of tuft cells in a scRNAseq analysis of the human airways using both bronchial brushing and endobronchial biopsy. This likely reflects the rarity of these cells and perhaps the methods used for the collection of airway material. Tuft cells express a range of indicative markers, including POU domain, class 2, transcription factor 3 (POU2F3), which is considered the lineagedefining transcription factor for tuft cell specification 40 , as well as transient receptor potential cation channel subfamily M member 5 (TRPM5). scRNAseq has now identified two terminally differentiated Trpm5 + tuft cell populations; one is positive for Gng13 and is likely to be responsible for 'taste' sensing, and the other is positive for Alox5ap, suggesting that it contributes to leukotriene synthesis 22, 23 . Tuft cells are thought to generate cysteinyl leukotrienes via the ATP sensor P2Y2 (ReF. 44 ). Although generally present in low numbers, the expansion of tuft cells in the airways occurs following the inhalation of common aeroallergens via activation of the cysteinyl leukotriene pathway. Endogenously generated leukotriene E 4 is sensed via CysLT 3 R, which regulates the number and function of murine airway brush cells 45 .

Thus, tuft cells are able to respond to a diverse range of signals via the combinatorial expression of dif ferent receptors. Tuft cells are thought to promote protective respiratory reflexes, such as sneezing, but can also con tribute to apnoea as well as to local neuro genic inflam mation of the mucosa 46, 47 . Given their distribution and function, tuft cells have also been referred to as 'soli tary chemosensory cells' , enhancing the chemorespon sive protection provided by the local neuronal system. scRNAseq analysis shows that mouse tuft cells express high levels of cholinergic and bitter taste signalling transcripts (Tas2r108, Gnat3, Trpm5). Acetylcholine is released from tracheal tuft cells following stimula tion with either the bitter taste receptor type 2 (TAS2R) ligand denatonium or Pseudomonas quinolone quorum sensing molecules, increasing mucociliary clearance mediated by TRPM5 and M3 muscarinic acetylcholine receptor (M3R), together with calcium release from cili ated cells 48 . It is postulated that the detection of these quorumsensing molecules from Gramnegative patho genic bacteria offers a mechanism by which the epithe lium triggers capsaicinsensitive nerve fibres, releasing calcitonin gene-related peptide (CGRP) and substance P, which promote innate immune cascades and microvas cular leak and combat bacterial invasion, thus limiting the population densities capable of forming destructive biofilms 43 .

Solitary chemosensory cells, with tuft cell properties, are found to be the primary epithelial source of IL25 in patients with chronic rhinosinusitis with nasal polyps, facilitating the activation of group 2 innate lymphoid cells (ILC2s) and the production of IL13, thereby maintain ing the type 2 environment in the upper airways 49 . Once activated, tuft cells can release neurotransmitters (such as acetylcholine), eicosanoids involved in leukotriene and prostaglandin biosynthesis and ATP, and cytokines such as IL25 and thymic stromal lymphopoietin (TSLP) 40, 42 . All of these mediators are implicated in aller gic inflammation, being able to elicit ILC2 activation and accumulation via the expression of specific receptors 50 .

Singlecell transcriptomic studies of the mouse tracheal epithelium and differentiated human tracheal epithelial cells in culture discovered a novel cluster of cells, termed 'pulmonary ionocytes' due to the similar ity of their gene expression profiles to those of the ion Box 2 | murine models of the airway epithelial niche the emphasis of this review is the human airway epithelial landscape. However, much of our understanding of the function of epithelial cells and their interaction with immune cells comes from experimental studies in laboratory mice. it is for this reason that we highlight salient proximal-distal variations in cellular composition and structural differences between the murine and human airway.

in mice, basal cells are largely restricted to the trachea and proximal bronchi yet, in humans, basal cells extend throughout the conducting airways 5, 6, 38, 140 . in humans, mucus-producing goblet cells are the most abundant secretory cell type in the proximal airways yet, in adult mice, these are comparatively rare and instead club cells predominate 5, 141 .

submucosal glands lie below the luminal surface and secrete mucus and other antimicrobial factors into the airway but also act as a stem cell niche containing myoepithelial basal cells 142, 143 . in mice, submucosal glands are localized to the larynx and the proximal trachea but, in humans, they are distributed throughout the cartilaginous airways.

the pseudostratified ciliated epithelium of the murine trachea is akin to that found throughout the human airways. therefore, it has been suggested that this may represent a more relevant region to study for translatable human airway epithelial cell biology 144 . in support of this concept, some of the single-cell findings from murine tracheal epithelial cells, including the discovery of the pulmonary ionocyte, have been recapitulated in studies of the human airway 19, 23 .

Beyond the trachea, the murine airway epithelium transitions to a simple columnar epithelium comprising ciliated, secretory (club) and clusters of pulmonary neuroendocrine cells, thus modelling the composition of only the most distal human airways 144 .

Mouse models are an indispensable tool for biomedical research and have revolutionized our understanding of immunology. However, it is noteworthy that important differences exist between the mouse and human immune system 145 . Differences in the living environment of mice, specifically microbial exposure history, have a profound influence on their immune cell repertoire 146, 147 . For example, in contrast to adult humans or pet-store mice, laboratory mice kept in specific-pathogen free facilities lack differentiated memory CD8 + T cell subsets 146 . these critical environmental differences profoundly affect the development of the pulmonary immune system and thus the interactions with the local epithelial landscape.

Turbinates small structures inside the nose that filter, warm and humidify air as it passes through the nostrils into the lungs.

Calcitonin gene-related peptide (CgRP) . A neuropeptide released from sensory nerve fibres.

www.nature.com/nri transport cells of the mucociliary epithelium of Xenopus larval skin 22, 23, 51 . As in Xenopus, ionocytes express genes encoding subunits of a VATPase proton pump, which regulates ion transport and pH. Crucial in determining the ionocyte lineage in mouse and human epithelial cells is FOXI1, which belongs to the forkhead family of transcription factors. Knockout studies in mice and lenti viral studies in human cell cultures show that FOXI1 regulates the expression of the CFTR gene that encodes a critical chlorideion transporter that is defective or absent in cystic fibrosis. Cystic fibrosis is a lifelimiting disease characterized by increased mucus viscosity, impaired mucociliary clearance, chronic infection and airway inflammation, leading to loss of lung function 52 . Ionocytes account for only 1-2% of human airway epi thelial cells but are more enriched in CFTR mRNA than in any other airway cell type. Combining scRNAseq with conventional in vivo lineage tracing over three different time points in a technique called 'pulseseq' , indicated that basal cells were the principal source of ionocytes and other rare cell types 23 . Given the clustering of rare cell types in scRNAseq data sets, Goldfarbmuren et al. further investigated rare cell lineage relationships using a CRISPR-Cas9 knockout system in human tracheal basal cells to determine that, during differentiation, ionocytes and pulmonary neuroendocrine cells (PNECs) arise from POU2F3 + tuftlike cells 26 .

PNECs are solitary cells resident within the surface epithelium of the trachea, bronchi and bronchioles. They can also exist in forma tions called neuroendocrine bodies in the intrapulmonary airways. PNECs function as chemosensors of the airway and respond to changes in oxygen, stretch and chemical stimuli 53 . They are a rich source of neuropeptides and neurotransmitters that elicit immune and physio logical effects. PNECs are the only innervated airway epithe lial cell type, are conserved across species and represent just 1% of the total lung epithelial cell population. They are reported to be the earliest specialized cell type that forms in the lung epithelium during development [53] [54] [55] and are thought to be particularly important function ally in early life since neonatal mice lacking PNECs are protected from allergic inflammation 56 . In vitro analyses predicted roles for PNECs in a range of activities, includ ing oxygen sensing, the maintenance of bronchial and vascular smooth muscle tone, and in the coordination of immune responses. However, an elegant series of experi ments with mutant mice has confirmed the functional characteristics of PNECs that influence a range of pulmo nary diseases. A defining characteristic marker of PNECs is the gene Ascl1, a transcription factor that is essential for their formation. Mice that lack Ascl1 die at birth, but a conditional knockout designed to inactivate Ascl1 in PNEC precursors led to viable mice that completely lack PNECs, as marked by a lack of CGRP + cells in the air way epithelium 56 . These Ascl1mutant mice were normal at baseline but were protected from developing severe goblet cell hyperplasia and type 2 inflammation when exposed to allergens during the perinatal period. This absence of PNECs was accompanied by a reduction in key neuropeptides, including the neurotransmitter gAbA.

The expression of the roundabout (ROBO) genes is vital for the clustering of PNECs into neuroendocrine bodies, limiting immune cell infiltration, and in pre venting alveolar simplification during postnatal lung development. Although PNEC defects were apparent in Robodeficient mice at E15.5, the physiological effects only occurred after birth, indicating that the effects of PNEC are dependent on the physical exposure to air that occurs with the first breath, thus reinforcing the idea that PNECs are sensors of the changing inhaled envi ronment. By contrast, the genetic ablation of PNECs in adult mice had no effect on airway homeostasis or repair following chemical injury.

In early life murine models, PNECs were shown to facilitate mucus hypersecretion during allergen expo sure via neurotrophin 4 regulation of PNEC innervation and the secretion of GABA, which promotes Muc5ac expression. PNECs represent the only source of GABA in primate lungs and in ex vivo cultures of human epi thelial cells 57, 58 . PNECs also reside in close association with ILC2s in the airways and communicate via CGRP to maximize the ILC expression of IL5 and GABA to elicit mucus production 56, 59 . GABA production was found to be absolutely required for goblet cell hyperpla sia but not in type 2 inflammation in both neonatal and adult models of allergic inflammation 56 . Importantly, these findings are reflected in humans, with increased numbers of the CGRP + subset of PNECs in patients with allergic asthma.

The fact that abnormalities in PNEC numbers are associated with a wide range of pulmonary diseases, including rare genetic disorders such as congenital dia phragmatic hernia and small cell lung cancer as well as more common diseases such as asthma, indicate a key role for these cells in physiological and immune path ways critical for effective functioning of the lung at homeostasis.

Immune properties of airway epithelial cells Antimicrobial mediators and airway mucins, includ ing MUC5AC and MUC5B, are produced by secre tory epithelial cells lining the airways and submucosal glands and contribute to the first layer of host defence at the airway epithelial surface 60 . Secretory IgA (SIgA)produced by subepithelial plasma cells and trans ported to the apical surface of airway epithelial cells via the polymeric immunoglobulin receptor (pIgR)prevents the adherence of airborne microorganisms in a process called 'immune exclusion' 61, 62 . Epithelial cells are equipped with pattern recognition receptors, such as Tolllike receptors, which rapidly sense and initiate an immune response to microbial threats, and cytokine receptors, including TNFR1, which allow them to respond to signals produced by immune cells such as airway macrophages 63 . Adjacent airway epithelial cells are linked by intracellular tight junctions and adherens junctions, which selectively regulate the paracellular diffusion of ions and molecules and maintain barrier integrity 64 . These tight junctions separate ligands present at the apical epithelial surface, for example, growth factor heregulin, from its ErbB family receptors located at the basolateral surface, so that activation occurs following GABA γ-aminobutyric acid, an inhibitory neurotransmitter.

specialized immune receptors that recognize conserved molecular structures on bacteria and viruses. These include Toll-like receptors. Nature reviews | Immunology injury and disruption of epithelial integrity 65 . Our view of the function of this mucosal surface has profoundly shifted because of studies demonstrating that airway epi thelial cells possess intricate and sophisticated properties that allow them to direct host immunity, inflammation and remodelling 66, 67 (Fig. 3 ).

The airway is an environment exposed to pollutants, pathogens and allergens known to induce apoptosis 68 . The clearance of apoptotic cells is carried out by 'professional' phago cytes, such as macrophages and dendritic cells 69 , but also by 'nonprofessional' phagocytes, including airway epithelial cells 70 . Apoptotic epithelial cells labelled with a CypHer5 dye that fluoresces within acidic phagolyso somes were directly engulfed through the recognition of phosphatidylserine by human bronchial epithelial cells (from a BEAS2B cell line) 70 . The inducible dele tion of the small GTPase RAC1, which is a downstream signalling molecule in the phagocytic engulfment path way, in the airway epithelium using a Ccsp-Cre/Rac1 fl/fl mouse model resulted in defective apoptotic cell phago cytosis and in a significant reduction in the production of the antiinflammatory cytokines transforming growth factorβ (TGFβ) and IL10 (ReF. 70 ). Critically, this translated into an exaggerated allergic airway inflammatory response in RAC1deficient mice, with elevated levels of epithelial cellderived IL33 detected in bronchoalveolar lavage (BAL) fluid 70 . This data supports the concept that airway epithelial cellmediated phago cytosis of apoptotic cells via RAC1 signalling plays a key role in taming the inflammatory response to common inhaled allergens. This study used a Ccsp-Cre mouse strain that specifically targets club cells and therefore this mechanism may not be generalizable to other types of airway epithelial cells.

The recognition of apoptotic cells by airway basal cells also plays a key role in determining cell pheno type and fate in the context of inflammation 66 . Basal cell hyperplasia is an early event in the pathogenesis of chronic obstructive pulmonary disease (COPD), an inflam matory lung disease 71 . The mechanism driving basal cell proliferation in the context of airway inflammation highlights a novel and previously unrecognized capabil ity of basal cells. Under homeostatic conditions, mouse tracheal basal cells express the AXL receptor (from the TAM receptor tyrosine kinase family) bound to GAS6 (a bridging molecule) 66 . In a mouse model of H1N1/PR8 influenza A virus infection, which is characterized by airway inflammation and ciliated epithelial cell apopto sis, basal cell reentry into the cell cycle and their prolif eration was promoted by the presence of AXL. Similarly, apoptotic thymocytes delivered intranasally to C57BL/6 mice increased the number of Ki67expressing prolifer ating basal cells, an effect that was significantly reduced in Axlknockout mice. Therefore, it has been inferred that basal cell proliferation during airway inflammation is mediated through the recognition of apoptotic cells by the AXL receptor tyrosine kinase 66 . In the absence of AXL, basal cells may differentiate into other cell types by socalled asymmetric cell division to acceler ate reepithelialization. Immunostaining of the small airway epithelium from human COPD tissue samples revealed that the number of AXLexpressing basal cells was increased and that their proliferation correlated with the presence of caspase 3positive apoptotic cells 66 .

Inflammatory memory. Not only are basal cells able to sense and respond to changes in the inflammatory microenvironment, but they also have an intrinsic capacity for inflammatory memory 72,73 -a discovery that was made in the context of a chronic allergic inflam matory disease 67 . Chronic rhinosinusitis(CRS) is a type 2 immunemediated disease characterized by inflamma tion and epithelial dysfunction in the nose and paranasal sinuses, with or without the formation of abnormal tis sue outgrowths called polyps 74 . Basal cell hyperplasia is a feature of tissue remodelling in this condition. Single cell transcriptomics was used to study human tissue samples collected during ethmoid sinus surgery from subjects with CRS with and without polyps as well as nasal scrapings from the inferior turbinate of healthy subjects and from those with CRS and polyps 67 showed the upregulation of transcription factors such as KLF5 and ATF3, known to maintain undifferentiated cell states, corresponding to enriched motifs in sorted polyp basal cells. These findings suggested that intrinsic changes at an epigenetic level may be responsible for the differences observed in polyp basal cell state and may be governed by the local type 2 inflammatory milieu. Bulk RNA sequencing of ex vivostimulated basal cells in submerged cultures revealed that IL4 and IL13 induced more than 10 times the number of genes in nonpolyp basal cells than in polyp basal cells. Additionally, at base line, the Wnt pathway activator CTNNB1 was expressed in polyp basal cells at levels only achievable in nonpolyp basal cells through IL4 and IL13 stimulation, suggest ing a 'memory' of in vivo exposure to type 2 cytokines by polyp basal cells. These data have profound impli cations for our understanding of allergic diseases given that efforts to treat these diseases are generally focused on manipulating cells of the immune system.

For over a decade, it has been recog nized that, in addition to neurohormonal control of the circadian rhythm by the suprachiasmatic nucleus in the brain, peripheral tissues and their cells, includ ing airway epithelial cells, possess an intrinsic circadian clock responsible for rhythmic immune oscillations with the time of day 75 . In mouse and human lung tissue, the clock gene products CLOCK and PER2 were expressed in CCSP + club cells 75 . Circadian oscillations, measured by the bioluminescence of PER2 in ex vivo mouse lung slices from transgenic mice expressing a Per2-luciferase fusion gene (Per2Luc mice), could be 'reset' in response to glucocorticoids and were lost with the selective abla tion of club cells. A notable timeofday variation in inflammatory response, in particular the BAL neutro phil count, was observed in response to an aerosolized lipopolysaccharide (LPS) challenge in C57BL/6 mice 76 . The targeted deletion of the clock gene Bmal1 in club cells using Ccsp-Bmal1 −/− mice augmented the duration of the neutrophilic inflammatory response to LPS chal lenge as well as to Streptococcus pneumoniae across the circadian cycle but without an increase in myeloper oxidase activity or a reduction in bacterial load 76 . Driving this pulmonary inflammatory response was the neutro phil chemoattractant CXCL5, which lost circadian regulation in the Bmal1 knockout. REVERBα is a nuclear receptor that sits in a nega tive feedback loop downstream of core transcriptional activators of the molecular clock, namely CLOCK and BMAL1 (ReF. 77 ). REVERBα plays a key role in the circa dian modulation of innate immune responses in mye loid cells and airway epithelial cells 78, 79 . The targeted deletion of the DNAbinding domain of REVERBα in CCSP + bronchial epithelial cells in mice led to the ampli fication of airway neutrophilic inflammation upon an aerosolized LPS challenge 79 . Again, this inflammatory response was mediated through the increased expres sion of CXCL5 at the transcript and protein level. Interestingly, the dual deletion of REVERBα and its paralog REVERBβ resulted in a further increase in neutrophilic inflammation upon LPS challenge in these mice as well as in unchallenged mice at steady state, suggesting that REVERB proteins play a homeostatic antiinflammatory function 79 . Inflammatory responses to LPS in vivo were not impacted by the deletion of REVERBβ alone, emphasizing the leading role of REVERBα in this pathway.

A further understanding of the local circadian control of cell function may reveal why some patients experience 'nighttime' asthma or cough.

Immune cell interactions at the epithelial surface scRNAseq facilitates the mapping of the diverse innate and adaptive immune cell communities of the airway niche at high resolution. Airway epithelial cells act in concert with resident and recruited immune cells to regulate pulmonary immunity (Fig. 4) . Whilst transcrip tomic studies of human sputum 80 and BAL 81,82 facilitate the detailed characterization of immune populations within the airway, bronchial brushing samples and biopsies capture both the epithelial and immune cell components 10 . Computational advances are now being leveraged to unveil the complex epithelial-immune crosstalk that occurs in the airway and alveolar space in health and disease 10, 83 .

Macrophages are the most abundant immune cells located in the airway lumen and are found readily in human sputum samples 80, 84 , airway brushings 10 and BAL 82, 85 . Murine studies show that resident airway macro phages are perinatally derived from fetal monocytes and selfmaintained locally throughout life 86, 87 . This paradigm was challenged by recent human work reveal ing, through scRNAseq, that following lung transplant, most tissueresident airway macrophages in the donor lung were replaced by airway macrophages from the cir culating monocyte pool of the recipient 88 . A singlecell transcriptomic study of sputum samples from patients with cystic fibrosis indicated a diseaserelated shift in dominant airway immune populations from resi dent airway macrophages to recruited monocytes and neutro phils, displaying proinflammatory phenotypes and gene expression changes suggestive of impaired phagocytosis 80 . Bidirectional interactions between resi dent airway macrophages and epithelial cells ensure the maintenance of a homeostatic state of immune tolerance to harmless stimuli and appropriate protective respon ses to inhaled pathogens with effective tissue repair when required 89 . A mouse model utilizing live confo cal microscopy showed that CD11cexpressing alveolar macrophages formed direct connections with alveolar epithelial cells by connexin 43containing gap junctions to limit the inflammatory response to LPS 90 . The explo ration of cell-cell signalling networks using scRNAseq data from lung tissue across four mammalian species (including humans) showed crosstalk between epithe lial cells and macrophages in the alveolar cell niche at homeostasis 83 .

Chronic obstructive pulmonary disease (CoPD). lung disease characterized by chronic airflow obstruction due to inflammation and remodelling of the small airways and destruction of the lung parenchyma.

Chronic rhinosinusitis (CRs). Type 2-mediated mucosal inflammation of the nose and paranasal sinuses. 

There are few studies that investigate epithelialmacrophage crosstalk in the human airways, yet this is a critically important area if we are to fully understand how dysregulated immunoregulatory interactions at this environmental interface contribute to human disease.

To explore the epithelial-immune axis in an inflam matory airways disease, BAL was studied in human sub jects with atopic asthma before and after subsegmental bronchoprovocation with an identified allergen 91 . Colonystimulating factor 1 (CSF1) was elevated in BAL samples after aeroallergen challenge and was shown to be secreted by airway epithelial cells. Epithelial cellspecific deletion of Csf1 in transgenic mice eliminated aller gic airway inflammation. Furthermore, epithelial cellderived CSF1 increased the numbers of a subset of alveolar dendritic cells expressing the CSFR1 receptor in BAL following allergen challenge and promoted the migration to regional lymph nodes 91 . Taken together, these findings suggest that epithelial cell-dendritic cell interactions in the airways enhance antigen presentation and augment adaptive allergic airway responses.

A robust immune response to respiratory patho gens at the airway epithelial surface is also contingent on an effective adaptive T cell response. Noteworthy is the induction of a subset of T cells -tissueresident memory T (T RM ) cells 92 -at the site of antigen entry in a spatially restricted niche around the airways, poised to generate a rapid immune response to subsequently www.nature.com/nri encountered pathogens 93, 94 . CD4 + and CD8 + T RM cells expressing CD69 were transcriptionally distinct to cir culating CD69effector memory T cells and expressed adhesion molecules that promote retention within the lung mucosal tissue 95 . Moreover, parabiosis experiments in influenzaimmune mice found that lung airway CD8 + T RM cells were continually replenished by inter stitial CD8 + T RM cells rather than by circulating mem ory T cells 96 . This was mediated by chemokine receptor CXCR6 binding to CXCL16 secreted by airway epithe lial cells and macrophages 96 . In human lung transplant recipients, donor T RM cells persisted in BAL samples from the lungs but not in peripheral blood for over 1 year 97 . A significant proportion of airway CD8 + T RM cells coexpressed the α E integrin CD103, which combines with β7 integrin to form the heterodimer α E β 7 and interacts with Ecadherin on epithelial cells 98 , promot ing retention at this mucosal site. Donor and recipient T cells were localized around the airways in biopsy sam ples and infiltrating T cells from the recipient acquired a T RM phenotype over time in BAL samples 97 . Crosstalk between T RM cells and airway epithelial cells can shape the immune response to environmental antigens. Pneumococcal pneumonia generates CD4 + T RM cells, which were discovered to finetune lung epi thelial cells to increase CXCL5 expression and enhance neutrophil recruitment during heterotypic recall infections 99 . In the context of a house dust mite allergic airways mouse model, the persistence of CD4 + T RM cells around the airways was associated with a rapid response to allergen rechallenge, resulting in airway hyperres ponsiveness, a cardinal feature of asthma 100 . Singlecell transcriptomics of airway wall biopsies from asthmatic patients and healthy controls defined two CD4 + T cell subsets -classic CD4 + T RM cells and a novel cluster called tissue migratory CD4 + T cells -that expressed genes associated with cell egression into tissues such as S1PR1 and SELL 10 . A bioinformatic tool was used in this study to interrogate cell-cell interactions in the asthmatic airway wall and unveiled increased predicted crosstalk between T helper 2 cells and epithelial cells, other immune cells, fibroblasts and smooth muscle cells known to play an important role in the pathogenesis of the disease.

The specific properties of the airway microenviron ment can drive adaptive gene expression changes in immune cells by epigenetic programming, which shape cell phenotype and function 101 . Compared with T RM cells in the spleen and lung interstitium, following influenza infection, CD8 + airway T RM cells displayed a distinct transcriptomic and epigenetic profile, enriched for genes associated with the integrated stress response and with amino acid starvation, which ultimately led to a gradual loss of these cells over time by apoptosis 101 .

Spanning the innate and adaptive arms of the immune system, a subset of innatelike T lymphocytes called mucosalassociated invariant T (MAIT) cells, contri bute approximately 4% of the total T cells in human air way wall biopsies 102 . The expression of a semiinvariant αβ T cell receptor (TCR) allows MAIT cells to recog nize metabolites of riboflavin (vitamin B 2 ) biosyn thesis derived from bacteria and yeasts and presented by MHCrelated protein 1 (MR1) 103 . When activated, MAIT cells generate a rapid proinflammatory cytokine response to provide protection against respiratory pathogens 104 but also display a tissue repair transcrip tomic signature, suggesting other important functions in barrier integrity and healing postinfection 105 .

Mediators produced by resident immune cells can hinder the epithelial cell response to pathogens at the airway mucosal surface. Type I (IFNα and IFNβ) and type III (IFNλ) interferons are key mediators in the host antiviral response but have recently been shown to compromise epithelial repair following infection 106, 107 . Sustained IFNλ produced by lungresident dendritic cells in response to polyinosine:polycytidylic acid (poly(I:C)), a synthetic viral RNA analogue, impaired epithelial barrier integrity, leading to severe pathology with Staphylococcus aureus superinfection in a mouse model 106 . During the recovery phase of murine influenza infection, airway epithelial proliferation and regeneration was reduced in a TP53dependent manner by prolonged exposure to IFNλ 107 . The nature of the epithelial inter feron response is also directly influenced by the type of invading pathogen. A delayed type I interferon response drives the accumulation of inflammatory monocytes and macrophages and leads to severe immunopathology in mice infected with SARSCoV 108 . Interestingly, the tran scriptional response of human bronchial epithelial cells to SARSCoV2 was found to be distinct from that induced by other respiratory viruses -genes associated with type I and type III interferon pathways were suppressed, yet there was a robust expression of genes encoding chemokines and proinflammatory cytokines, which is likely to be responsible for many of the clinical manifes tations of the disease 109 . Defects in the type I interferon response due to rare genetic variants or IgG neutralizing autoantibodies were also discovered in a proportion of patients with severe COVID19 pneumonia 110, 111 .

Complex interactions exist between epithelial cells and innate and adaptive immune cells in the airway to maintain host defence. Signals from epithelial cells maintain tissue resident immune cells and modulate their response to antigen while, conversely, immune cells can directly alter the function and phenotype of airway epithelial cells.

The general location of airway epithelial cells enables them to sense and react to environmental change via the secretion of a range of mediators that facilitate interac tions with immune and stromal cells in the underlying parenchyma. In addition to the specialized epithelial cells that monitor the airways and trigger avoidance reflexes, such as coughing and sneezing, it is well recog nized that the airways are richly innervated with sensory neurons, facilitating the execution of these avoidance reflexes 112 . Recent evidence indicates that extensive crosstalk between neuronal and immune systems and specialized chemosensory pulmonary epithelial cells is vital for the regulation of tissue homeostasis 113, 114 (Fig. 5 ). This integrated landscape is also able to coordinate responses to invading patho gens or noxious particles, to monitor environmental changes such as fluctuations in temperature or oxygen, or even to react to physical Parabiosis surgical procedure in which two animals are joined so that they share the same circulation.

Nature reviews | Immunology events such as stretch or constriction that occur dur ing gasping. For example, PIEZO1, a mechanosensory ion channel expressed by myeloid cells in mice, was shown to sense environmental cyclical hydrostatic pressure changes and generate a proinflammatory response 115 .

The lungs contain a range of different neural cells, including nociceptor sensory neurons that react to noxi ous or potentially harmful stimuli, while cholinergic neurons regulate airway tone, airway smooth muscle contraction, mucus secretion and vasodilation via the interaction with muscarinic acetylcholine receptors (mAChRs) found on airway smooth muscle, glands and pulmonary vasculature. Given that many basic respira tory responses, such as cough, are mediated by sensory neurons 116 , it is perhaps not surprising that they are pre dicted to play an important role in lung inflammatory responses. In addition to relaying sensory changes to the brain, activated lung neurons can themselves release stimulatory peptides that contribute to the ensuing inflammatory reaction. Direct evidence of their contri bution to pulmonary inflammation stems from exper iments in which the ablation or chemical suppression of Nav1.8 + or transient receptor potential vanilloid type 1 (TRPV1) sensory neurons resulted in reduced allergic inflammation and bronchial hyperresponsiveness [117] [118] [119] . Similarly, targeted lung denervation, a bronchoscopic radiofrequency ablation therapy, is used in patients to durably disrupt parasympathetic pulmonary nerves because it has been found to decrease airway resistance, mucus hypersecretion and inflammation 117, 120 in patients with COPD.

The combination of specialized chemosensory cells and direct neuronal interaction may also represent an alternative way in which the airways respond to inhaled respiratory pathogens, airborne pollutants and aller gens. The activation of epithelial TRPV4 triggers pro tective responses to bacterial LPS, an increasing ciliary Transient receptor potential vanilloid type 1 (TRPV1). Non-selective cation channel activated by diverse stimuli, including capsaicin, heat and low pH, and expressed on a variety of cell types. Pulmonary reactions to stimuli, such as pathogens, pollutants, toxins and environmental changes, result in avoidance reflexes and inflammation. Sensory neurons release vasoactive peptide (VIP), which binds to VIP receptor 2 (VIPR2) expressed by group 2 innate lymphoid cells (ILCs) and T helper 2 cells, resulting in the release of IL-5, which stimulates the release of more IL-5 from these neurons, generating an amplifying loop to enhance allergic responses. Communication between group 2 ILCs and pulmonary neuroendocrine cells (PNECs) occurs via calcitonin gene-related peptide (CGRP) to maximize the ILC-derived expression of IL-5 and GABA to elicit mucus generation. By contrast, CGRP inhibits neutrophils and γδ T cells during bacterial infections. Cholinergic neurons react to allergens and helminths to amplify type 2 inflammation via the secretion of neuromedin U (NMU) which reacts with NMU receptor 1 (NMUR1) on group 2 ILCs to enhance cytokine secretion.

www.nature.com/nri beat frequency and the production of bactericidal nitric oxide. Mice lacking TRPV4 display exacerbated airway hyperreactivity and enhanced neutrophil recruitment into the airways 121 . Conversely, the specific deletion of TRPV1 + neurons resulted in enhanced immune pro tection to lethal pneumonia induced by S. aureus via increased survival cytokine induction and bacterial clearance 122 . TRPV1 and Nav1.8 + nociceptor sensory neurons were shown to influence bacterial dissemi nation via the suppression of neutrophil numbers and surveillance activities and the regulation of resident γδT cell numbers. The specific ablation of vagal TRPV1 neu rons resulted in enhanced antibacterial immunity via the release of the neuropeptide CGRP. Epithelial-neuronal interactions facilitate lung inflammation via the secretion of neuropeptides and neurotransmitters. Many immune cells express recep tors that facilitate communication with the epithelium but also directly with nerves 123 . The discovery that ILC2s express a range of receptors for neuropeptides and neuro transmitters reveals a mechanism by which neuronal and immune systems integrate to promote a range of type 2 cytokine responses that facilitate antimicrobial, inflam matory and tissue protective type 2 repair responses at mucosal sites 124 . ILC2 residency in the airways is regu lated by signals delivered systemically or by the local tis sue environment 125 and these cells are enriched at branch points, similar to PNECs 56 . ILC2s interact directly with neurons via neuropeptides, neurotransmitters and neuro trophic factors, including CGRP, neuromedin U (NMU), acetylcholine and vasoactive intestinal peptide (VIP) and such interactions facilitate both stimulatory and inhib itory signals 123 . ILC2 expression of VIPR2 enables the cells to respond to neuronalderived VIP by secreting IL5, thus creating a feedback loop whereby IL5 stim ulates nociceptors to produce more VIP. NMUR1 sig nalling amplifies ILC2driven allergic inflammation 126 . NMU secreted by thoracic dorsal root ganglia interacts with NMUR1 expressed by lung ILC2s and regulated by IL25. The loss of NMUR1 reduces ILC2 responses and type 2 inflammation 127 . NMU also promotes the con traction of smooth muscle cells, although this has not been shown specifically in pulmonary smooth muscle cells 128 . Conversely, CGRP has differential effects on the immune response to allergeninduced inflammation. Mice lacking PNECs have a reduced expression of both CGRP and GABA after allergen exposure. CGRP induces IL5 secretion from ILC2s and inactivation of the CGRP receptor (Calcr1) in ILC2s in vitro ameliorates allergic inflammation 56 . CGRP is also found to negatively regu late type 2 responses to either allergen exposure or hel minth infection 59, 126 . CGRP modulates type 2 cytokine production from ILC2s that have been activated by alarmins or NMU, thereby constraining the magnitude of the type 2 responses in vivo 59 . PNECderived GABA seems to affect mucus secretion since the inactivation of GABA production results in defective goblet cell hyper plasia and direct instillation of GABA in PNECdeficient mice restores mucus production 56 . These differential effects of neuropeptides on ILC2 cytokine production and type 2 immune pathology, concomitant with epithe lial cellderived alarmins and neuropeptides, provides an integrated mechanism whereby tissue responses to allergen can be finetuned according to changing circumstances.

Our views of the human airway epithelium have shifted dramatically, catalysed by the advancement in techno l ogies that facilitate singlecell transcriptomics. The cel lular communities of the airway epithelium are diverse, dynamic and yet functionally cohesive. Epithelial cells work collaboratively with localized populations of immune cells to support a multifaceted frontline defence system with highly effective injuryrepair responses.

Epithelial, stromal and immune cells in the lung can be modulated by many aspects of their local tissue microenvironment, including the properties of the extra cellular matrix 129, 130 , mechanical stress 131 and exposures such as to cigarette smoke 26, 132 . Mapping cell location in a specific anatomical tissue microenvironment using spatial transcriptomic techniques 133 will undoubtedly enhance our understanding of cell molecular phenotype and function within a particular spatial niche.

A multiomic approach utilizing singlecell tran scriptomics and mass spectrometrybased proteomics was used to chart the molecular and cellular alterations that occur in the lung with ageing 134 . This work showed alterations in airway epithelial cell composition and lung extracellular matrix with increasing age. The application of a similar approach to human tissue samples in com bination with comprehensive demographic and clinical information will allow us to link changes in cell biology with disease phenotype.

The functional validation of transcriptomic data that has been generated through complex bioinformatics requires robust in vitro culture systems 135 . The human 'smallairwayonachip' was developed to recapitulate the airway tissue microenvironment and cell responses in health and disease, and represents a significant devel opment in this area 136 . This system comprises a micro fluidic device with a fully differentiated, mucociliary airway epithelium cultured adjacent to a microvascular endothelium in a channel exposed to media under flow. It has been used to model inflammatory airway diseases and study the interplay between epithelial cells and immune cells in vitro.

The airway epithelium is dynamic and continuously replenished by basal progenitor cells 38, 137 . Singlecell transcriptomics has been used to elucidate airway basal cell differentiation and response to injury in vitro 24 . Fascinating mechanisms exist to preserve a healthy barrier in other stratified epithelial surfaces around the body such as the skin. A natural phenomenon of cell competition was observed to operate in a mouse model of skin development -in a mosaic epidermal tissue, socalled 'winner' progenitor cells induce apoptosis in neighbouring apoptotic 'loser' progenitor cells and eliminate them by engulfment 138 . Cell losers not cleared by this mechanism were later eliminated from the basal cell layer by differentiation through the stratified cell layer. This is physiologically important because, when cell competition was disturbed, epithelial barrier func tion was compromised 138 . Whether this competitive Neuromedin U (NMU). A neuropeptide with a diverse range of functions, including smooth muscle contraction, pain perception, regulation of blood pressure, stress responses and feeding behaviour.

Vasoactive intestinal peptide (ViP). A neuropeptide.

Nature reviews | Immunology cell behaviour is conserved throughout other epithe lial tissues, including the airway lining, remains to be elucidated and warrants further investigation.

Transcriptomic technologies have enhanced our view of the cellular landscape in the human lung but there remain inherent limitations and many unans wered questions. Loss of fragile cell types in the pro cess of sequencing can bias the analysis and variation in bioinformatic pipelines and cell annotation can hinder comparisons between different studies. Rare epithelial cell types have attracted much interest but, given their low frequency within the lung, the challenge will be to ascertain their contribution to diverse disease patho l ogies in the hope that we can harness and manipulate particular cells to promote lung health across the human life course.

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Early local immune defences in the respiratory tract

Ultrastructure and function of the human tracheal mucosa

The cells of the pulmonary airways

Early studies on the surface epithelium of mammalian airways

Cell number and distribution in human and rat airways

Number and proliferation of basal and parabasal cells in normal human airway epithelium

Number and proliferation of clara cells in normal human airway epithelium

The respiratory tract microbiome and lung inflammation: a two-way street

The cellular and physiological basis for lung repair and regeneration: past, present, and future

This was the first study to use scRNA-seq to analyse airway brushing and biopsy samples from human subjects

Computational methods for single-cell RNA sequencing

Benchmarking single-cell RNA-sequencing protocols for cell atlas projects

This review provides a step-by-step discussion of the elements that comprise a typical scRNA-seq analysis workflow

The human lung cell atlas: a high-resolution reference map of the human lung in health and disease

Single-cell reconstruction of the early maternal-fetal interface in humans

COVID-19 severity correlates with airway epithelium-immune cell interactions identified by single-cell analysis

CellPhoneDB: inferring cell-cell communication from combined expression of multisubunit ligand-receptor complexes

Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis

A single-cell atlas of the human healthy airways

This study used scRNA-seq to profile airway epithelial cell types from brushings and biopsies taken at distinct locations throughout the nose, trachea and bronchi of healthy volunteers

scRNA-seq assessment of the human lung, spleen, and esophagus tissue stability after cold preservation

Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations

This study used scRNA-seq to interrogate the gene expression profiles of mouse tracheal and cultured human bronchial epithelial cells, delineated the differentiation trajectories during homeostasis and tissue repair

A revised airway epithelial hierarchy includes CFTR-expressing ionocytes

This seminal study utilized scRNA-seq to expose airway epithelial cell diversity and refine lineage hierarchies in the mouse trachea, notably discovering 'hillock' structures and the 'ionocyte

Novel dynamics of human mucociliary differentiation revealed by single-cell RNA sequencing of nasal epithelial cultures

CDC20B is required for deuterosome-mediated centriole production in multiciliated cells

This study used scRNA-seq to evaluate the impact of cigarette smoking on each airway cell type in the human tracheal epithelium

Single-cell RNA-seq reveals ectopic and aberrant lung-resident cell populations in idiopathic pulmonary fibrosis

Single-cell RNA sequencing reveals profibrotic roles of distinct epithelial and mesenchymal lineages in pulmonary fibrosis

Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq

Using single-cell RNA sequencing to unravel cell lineage relationships in the respiratory tract

The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells

This study evaluated the performance of 45 trajectory inference methods on different data sets and devised practical guidelines for their use

RNA velocity of single cells

Generalizing RNA velocity to transient cell states through dynamical modeling

Diffusion maps for high-dimensional single-cell analysis of differentiation data

SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes

SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues

Basal cells as stem cells of the mouse trachea and human airway epithelium

Clonal dynamics reveal two distinct populations of basal cells in slow-turnover airway epithelium

Regulation of immune responses by tuft cells

Detection of succinate by intestinal tuft cells triggers a type 2 innate immune circuit

Tuft-cell-derived IL-25 regulates an intestinal ILC2-epithelial response circuit

This article shows how chemosensory cells in the airways mediate reflex reactions to irritants, including bacterial quorum-sensing molecules, describing a mechanism by which bacterial population densities are limited by the local epithelial inflammatory response

Airway brush cells generate cysteinyl leukotrienes through the ATP sensor P2Y2

The cysteinyl leukotriene 3 receptor regulates expansion of IL-25-producing airway brush cells leading to type 2 inflammation

Cholinergic chemosensory cells in the trachea regulate breathing

Tuft cells-systemically dispersed sensory epithelia integrating immune and neural circuitry

Tracheal brush cells release acetylcholine in response to bitter tastants for paracrine and autocrine signaling

Solitary chemosensory cells are a primary epithelial source of IL-25 in patients with chronic rhinosinusitis with nasal polyps

A metabolite-triggered tuft cell-ILC2 circuit drives small intestinal remodeling

Specification of ion transport cells in the Xenopus larval skin

This article describes how PNECs act as rheostats to enable the lungs to sense and respond to changes in the inhaled environment

Functional facets of the pulmonary neuroendocrine system

Functional characterization of pulmonary neuroendocrine cells in lung development, injury, and tumorigenesis

Pulmonary neuroendocrine cells amplify allergic asthma responses

Pulmonary neuroendocrine cells secrete γ-aminobutyric acid to induce goblet cell hyperplasia in primate models

Early life allergen-induced mucus overproduction requires augmented neural stimulation of pulmonary neuroendocrine cell secretion

Neuropeptide CGRP limits group 2 innate lymphoid cell responses and constrains type 2 inflammation

Airway mucus function and dysfunction

Regulation of the polymeric immunoglobulin receptor and IgA transport: new advances in environmental factors that stimulate pIgR expression and its role in mucosal immunity

Airway bacteria drive a progressive COPD-like phenotype in mice with polymeric immunoglobulin receptor deficiency

Differential regulation of the transcriptomic and secretomic landscape of sensor and effector functions of human airway epithelial cells

Pulmonary epithelial barrier function: some new players and mechanisms

Segregation of receptor and ligand regulates activation of epithelial growth factor receptor

Sensing of apoptotic cells through Axl causes lung basal cell proliferation in inflammatory diseases

Allergic inflammatory memory in human respiratory epithelial progenitor cells

This study demonstrates that basal stem cells in the human nasal epithelium acquire and retain allergic memory, contributing to chronic inflammatory disease

Apoptosis and the airway epithelium

Apoptotic cell removal in development and tissue homeostasis

Apoptotic cell clearance by bronchial epithelial cells critically influences airway inflammation

Early events in the pathogenesis of chronic obstructive pulmonary disease. Smoking-induced reprogramming of airway epithelial basal progenitor cells

Adaptation and memory in immune responses

Distribution and storage of inflammatory memory in barrier tissues

Immunopathogenesis of chronic rhinosinusitis and nasal polyposis

Circadian timing in the lung; a specific role for bronchiolar epithelial cells

An epithelial circadian clock controls pulmonary inflammation and glucocorticoid action

Immunity around the clock

The nuclear receptor REV-ERBα mediates circadian regulation of innate immunity through selective regulation of inflammatory cytokines

Circadian clock component REV-ERBalpha controls homeostatic regulation of pulmonary inflammation

Single cell transcriptional archetypes of airway inflammation in cystic fibrosis

Proliferating SPP1/MERTK-expressing macrophages in idiopathic pulmonary fibrosis

Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19

Single-cell connectomic analysis of adult mammalian lungs

Non-invasive analysis of the sputum transcriptome discriminates clinical phenotypes of asthma

Flow cytometric analysis of myeloid cells in human blood, bronchoalveolar lavage, and lung tissues

Alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via GM-CSF

Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes

Dynamics of human monocytes and airway macrophages during healthy aging and after transplant

Airway macrophages as the guardians of tissue repair in the lung

Sessile alveolar macrophages communicate with alveolar epithelium to modulate immunity

Airway epithelial cell-derived colony stimulating factor-1 promotes allergen sensitization

Tissue-resident T cells and other resident leukocytes

Lung niches for the generation and maintenance of tissue-resident memory T cells

Trigger-happy resident memory CD4 + T cells inhabit the human lungs

Human tissue-resident memory T cells are defined by core transcriptional and functional signatures in lymphoid and mucosal sites

Interstitial-resident memory CD8 + T cells sustain frontline epithelial memory in the lung

Generation and persistence of human tissue-resident memory T cells in lung transplantation

Direct and regulated interaction of integrin αEβ7 with E-cadherin

Lung CD4 + resident memory T cells remodel epithelial responses to accelerate neutrophil recruitment during pneumonia

Biased generation and in situ activation of lung tissue-resident memory CD4 T cells in the pathogenesis of allergic asthma

Environmental cues regulate epigenetic reprogramming of airway-resident memory CD8 + T cells

Steroid-induced deficiency of mucosal-associated invariant T cells in the chronic obstructive pulmonary disease lung. Implications for nontypeable haemophilus influenzae infection

Mucosal-associated invariant T cells and disease

MAIT cells protect against pulmonary Legionella longbeachae infection

Activation and in vivo evolution of the MAIT cell transcriptome in mice and humans reveals tissue repair functionality

Type III interferons disrupt the lung epithelial barrier upon viral recognition

Type I and III interferons disrupt lung epithelial repair during recovery from viral infection

Dysregulated type I interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in SARS-CoVinfected mice

Imbalanced host response to SARS-CoV-2 drives development of COVID-19

Inborn errors of type I IFN immunity in patients with life-threatening COVID-19

Auto-antibodies against type I IFNs in patients with life-threatening COVID-19

An airway protection program revealed by sweeping genetic control of vagal afferents

The regulation of immunological processes by peripheral neurons in homeostasis and disease

Neuro-immune cell units: a new paradigm in physiology

Mechanosensation of cyclical force by PIEZO1 is essential for innate immunity

This article describes how mechanical forces influence innate immune pathways in the lungs via the expression of PIEZO1, a mechanically activated ion channel

Afferent pathways involved in reflex regulation of airway smooth muscle

Safety and adverse events after targeted lung denervation for symptomatic moderate to severe chronic obstructive pulmonary disease (AIRFLOW). A multicenter randomized controlled clinical trial

Silencing nociceptor neurons reduces allergic airway inflammation

Population of sensory neurons essential for asthmatic hyperreactivity of inflamed airways

Anti-inflammatory effects of targeted lung denervation in patients with COPD

TRPV4 activation triggers protective responses to bacterial lipopolysaccharides in airway epithelial cells

Nociceptor sensory neurons suppress neutrophil and γδ T cell responses in bacterial lung infections and lethal pneumonia

This study shows how lung sensory neurons contribute to innate host defence mechanisms against lethal bacterial infections

Neuronal regulation of innate lymphoid cells

Neuroimmune circuits in inter-organ communication

ILC2s -resident lymphocytes pre-adapted to a specific tissue or migratory effectors that adapt to where they move?

The neuropeptide NMU amplifies ILC2-driven allergic lung inflammation

The neuropeptide neuromedin U stimulates innate lymphoid cells and type 2 inflammation

Species-dependent smooth muscle contraction to Neuromedin U and determination of the receptor subtypes mediating contraction using NMU1 receptor knockout mice

Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans

Mechanosensing by the alpha6-integrin confers an invasive fibroblast phenotype and mediates lung fibrosis

Progressive pulmonary fibrosis is caused by elevated mechanical tension on alveolar stem cells

Characterizing smoking-induced transcriptional heterogeneity in the human bronchial epithelium at single-cell resolution

Slide-seq: a scalable technology for measuring genome-wide expression at high spatial resolution

An atlas of the aging lung mapped by single cell transcriptomics and deep tissue proteomics

Airway and alveolar epithelial cells in culture

Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro

Injury induces direct lineage segregation of functionally distinct airway basal stem/progenitor cell subpopulations

Distinct modes of cell competition shape mammalian tissue morphogenesis

Resolving the fibrotic niche of human liver cirrhosis at single-cell level

Immunohistochemical and ultrastructural studies of basal cells, Clara cells and bronchiolar cuboidal cells in normal human airways

The distribution and structure of cells in the tracheal epithelium of the mouse

The glandular stem/ progenitor cell niche in airway development and repair

Myoepithelial cells of submucosal glands can function as reserve stem cells to regenerate airways after injury

Airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling

Making mouse models that reflect human immune responses

Normalizing the environment recapitulates adult human immune traits in laboratory mice

The comparative immunology of wild and laboratory mice, Mus musculus domesticus

CML is a Wellcome Senior Fellow in Basic Biomedical Sciences (107059/z/15/z), RJH is funded through the Imperial College Clinician-Investigator Scholarship programme.

The authors contributed equally to all aspects of the article.

The authors declare no competing interests.

Nature Reviews Immunology thanks J. Kolls and J. Ordovas-Montanes for their contribution to the peer review of this work.

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