Regulation of Immune Responses by Nonhematopoietic Cells in Asthma

The lung is a highly branched organ that functions primarily to allow uptake of oxygen and removal of carbon dioxide. However, this task comes with inherent danger, as inhaled air contains a diverse array of environmental agents, including pollutants, allergens, and pathogens. The lung must have the capacity to mount effective responses to pathogens and toxins, but inappropriate or overexuberant immune responses in the lung can also threaten health by compromising gas exchange, as occurs in a plethora of diseases, including COVID-19. Inappropriate immune responses are also at the core of asthma, a widespread, chronic airway disease, characterized by intermittent dyspnea (shortness of breath) due to airway inflammation, excessive mucus production, and airway hyperresponsiveness (AHR) (1). Approximately 8% of adults in the U.S. currently have asthma, and the annual, nationwide cost of this disease is enormous (2).

For many years, asthma was regarded as a single disease, but it is now seen as a spectrum of lung pathologies with overlapping features. The relatively new concept of asthma endotypes postulates that different forms of asthma arise from perturbations of distinct molecular and cellular pathways (3). The most common and best-characterized form of this disease is allergic asthma. Individuals with this form of disease display eosinophilic airway inflammation, elevated allergen-specific IgE titers, and generally respond well to treatment with inhaled or oral glucocorticoids (4, 5). However, prolonged glucocorticoid use is associated with several adverse effects (reviewed in Ref. 6). Furthermore, approximately half of asthmatic patients display noneosinophilic forms of disease. These patients often exhibit neutrophilic airway inflammation and are notoriously resistant to inhaled corticosteroids (4). An improved understanding of the molecular and cellular basis of different asthma endotypes should reveal novel pathways that can be selectively targeted in specific forms of asthma.

The immune cells and cytokines that drive allergic airway disease have been studied for many years and are well characterized (reviewed in Ref. 7). However, emerging evidence also points to nonhematopoietic cells as unindicted coconspirators in the pathogenesis of asthma with untapped potential for therapeutic intervention. Thus, although nonhematopoietic cells are integral to gas exchange and structural integrity, they also contribute to local inflammatory responses and host defense. In this review, we summarize recent advances in our understanding of how nonhematopoietic cells, particularly airway epithelial cells (AECs), can alter immune cell function in the lung and contribute to asthma. Finally, we briefly highlight emerging evidence for the roles of other lung-resident nonhematopoietic cell populations in asthma pathogenesis.

Allergic responses that lead ultimately to asthma begin when inhaled allergens are captured by lung dendritic cells (DCs), which then move to regional lymph nodes to instruct the differentiation of allergen-specific naive T cells to effector Th2 lymphocytes. These cells produce IL-4, IL-5, and IL-13, thereby promoting Ab class switching to IgE, eosinophilic inflammation, and AHR, respectively (8, 9). This allergic form of asthma is sometimes referred to as type 2 (T2)–high. By contrast, T2-low forms of disease are less well understood, in part because of their heterogeneity, which includes neutrophilic, mixed granulocytic, and paucigranulocytic forms of asthma (10, 11). Indeed, T2-low asthma is defined primarily as lacking T2 pathway signatures (12). T2-low asthma is often severe and associated with a relatively late age of onset and obesity (13). It is likely that multiple pathways including Th17 cells contribute to T2-low asthma. Evidence in support of Th17-mediated neutrophilia in T2-low asthma has primarily emerged from preclinical animal studies, but at least some human patients within this broad category have IL-17–producing Th17 cells that promote neutrophilia (14–16). Notably, Th17 cells are relatively resistant to corticosteroids (17), which might at least partly account for the steroid-resistant nature of neutrophilic asthma. In addition to promoting neutrophil chemotaxis (17–23), IL-17 also enhances methacholine-induced tracheal ring contraction and airway narrowing (24). Although blockade of IL-17 signaling in a clinical trial had equivocal outcomes for asthmatic patients, cohort selection and stratification were based upon treatment-refractory disease and not a pertinent phenotype (e.g., IL-17–high or neutrophilic asthma) (25). Recent advances in cutting edge technologies, such as single-cell RNA sequencing (scRNA-seq), will continue to expand our knowledge of how various leukocytes and nonhematopoietic cells initiate and maintain different forms of T2-low asthma (26). This new knowledge should reveal novel pathways that can be selectively targeted in novel therapies to treat specific T2-low subtypes (Fig. 1).

T2 innate lymphoid cells (ILC2s) are also important mediators of allergic disease, including asthma. In addition to producing large amounts of IL-5 and IL-13, ILC2s cross-regulate conventional T cells and promote tissue repair in the lung through production of amphiregulin (AREG) (27, 28). ILC2s are more abundant in the blood and sputum of severe asthmatics than in those of mild asthmatics (29). Moreover, ILC2 frequency and activation status are enhanced in patients undergoing glucocorticoid therapy (30), suggesting that ILC2s might contribute to the severity of steroid-resistant, T2-high asthma.

The developmental pathways for effector T cells and ILC2s have been largely extrapolated from cell culture experiments to which various combinations of cytokines have been added. Anti-CD3/anti-CD28 agonist Abs are often used to artificially mimic the actions of Ag-presenting DCs. Although many in vitro studies of T cell differentiation have used DCs, they are often obtained from the spleen or derived from bone marrow (BM) cells. Such studies lack contextual cues from the local microenvironment of the lung, including nonhematopoietic structural cells (Fig. 2A), which recent studies show are critically important in shaping organ-specific immunity (31). Cell culture experiments that include primary nonhematopoietic lung cells should prove useful in dissecting their contributions to immune cell activation.

Animal models offer the ability to manipulate genetic and environmental contributions to allergic inflammation of the airway. As with human asthma, most animal models of this disease comprise two phases: an asymptomatic sensitization phase in which allergen presentation by lung DCs to T cells begins a maladaptive immune response and an allergen challenge phase that elicits many features of allergic asthma. In most current models of asthma, animals are sensitized by administering allergens directly to the airway, an approach that mimics the presumptive route of human exposure to airborne allergens. These models reproduce a complex pattern of leukocyte trafficking and activation that includes DCs, macrophages, T cells, eosinophils, and neutrophils. Much of this response is dependent on signals provided by nonhematopoietic lung-resident cells.

Although mice are used extensively for preclinical asthma studies, important differences exist between human and mouse lungs at both the anatomical and cellular levels, and these differences have pragmatic implications for translational research (32, 33). In particular, the conducting airways of the mouse quickly branch into alveolar ducts, which may result in greater inflammation within the alveoli in mouse models of asthma. Human lungs, in contrast, exhibit more pronounced branching within the bronchioles, and allergic asthma is generally thought to be limited to the conducting airways. However, some clinical evidence suggests that alveolar inflammation in humans can impact lung function in at least some forms of asthma (34). Thus, although there are many parallels between human asthma and mouse models of allergic airway disease, some caution should be taken in extrapolating mouse data to human asthma. Nonetheless, nonhematopoietic cells, such as AECs, of both species share multiple physical and functional features, and mouse studies have the potential to provide valuable insights into the cellular and molecular underpinnings of human asthma.

Nonhematopoietic pulmonary cells respond to environmental stimuli and alert the host to environmental change, in part through their production of cytokines (recently reviewed in Ref. 35). Thus, a comprehensive understanding of pulmonary immunity requires knowledge of how environmental changes are detected by nonhematopoietic cells, the signaling molecules these cells produce, and how they affect downstream immune pathways. As the role of epithelial cells (ECs) in allergic airway disease is much better characterized than that of the mesodermal, endothelial, or neural components, we focus in this review primarily on ECs.

Although the lung contains at least 40 different cell types (36), it is composed of two basic functional units: 1) the alveoli, where gas exchange occurs and 2) the conducting airways. The entire internal surface of the lung is lined by ECs that provide a barrier between potentially harmful components of inhaled air and the underlying tissue. Although this barrier function is common to all ECs, their morphology and functions differ profoundly in different parts of the lung (37). Elegant lineage-tracing studies in the mouse have proven useful in understanding the development of various types of ECs at homeostasis and after injury (38, 39).

The luminal surface of alveoli are primarily lined by a mosaic of alveolar type 1 cells (AT1). The thin, flat nature of AT1 cells maximizes gas exchange between CO₂-laden blood in pulmonary capillaries and inhaled O₂. A second alveolar EC known as alveolar type 2 cells (AT2) have distinct functions, including the production of surfactants that prevent collapse of the alveoli during exhalation. AT2 cells also serve as progenitors of type 1 cells (38) and secrete an array of molecules, including surfactants and defensins that contribute to host defense. Of specific relevance to airway inflammation, AT2 cells produce the potent T2 innate cytokine IL-33 in mouse models of asthma (40) and in human chronic obstructive pulmonary disease (41). This IL-33 production by AT2 cells can act on basophils to reprogram macrophages in the mouse (42). It is possible that this is related to the observed role of alveolar inflammation in at least some forms of asthma (34), but alveoli are anatomically distant from the affected airways, and the role of cytokine production by AT2 cells in human asthma remains uncertain.

Like alveoli, the conducting airways of the lung are also lined with ECs, but these cells are quite distinct from either AT1 or AT2 cells. AECs function collectively to sense and respond to changes in the environment, remove toxins and debris, provide resistance to infection, and repair and repopulate damaged cells. In addition to their function as a physical barrier to pathogens and toxins, ECs participate in innate defense through secretion of microbicidal molecules and mucus that trap and kill noxious agents. ECs are also critical to host immunity and respond to infection by increased expression of pattern recognition receptors, such as TLR2 and TLR4 (43), and protease-activated receptor-2 (PAR-2). Activation of these receptors triggers release of an array of cytokines that have wide-ranging effects on immune responses. The most common cells in the airway epithelium are ciliated cells, club cells, goblet cells, and basal cells (Fig. 2).

The large and medium airways contain large numbers of multiciliated ECs whose primary role is to continually move debris- and pathogen-trapping mucus in an escalator-like fashion from the airways toward the pharynx. Transcriptionally, these cells are defined by the presence of the transcription factor, FoxJ1. They are terminally differentiated cells that can arise from secretory cells or from basal cells, depending on their position in the lung and whether the host is at steady-state or recovering from injury (44).

There are two types of secretory cells: club cells and goblet cells. Club cells are abundant, nonciliated, columnar cells whose cytoplasm is filled with secretory granules containing antimicrobial and anti-inflammatory proteins (45). Interestingly, although secretory cells are “fully differentiated” in terms of their primary function, lineage-tracing studies indicate that these cells also retain the capacity to self-renew and to differentiate into ciliated cells in the airway (46). Goblet cells synthesize the mucins Muc5AB and Muc5AC, which are stored in vesicles. The numbers of these cells can undergo dramatic expansion during allergic airway inflammation, and like ciliated cells, can also arise from club cells, particularly in the presence of IL-13.

Basal cells are characterized morphologically by their small height compared with their neighboring luminal ECs and functionally by their stem cell–like capacities for self-renewal and their ability to serve as progenitors of multiple EC subtypes, including secretory cells, ciliated cells, and neuroendocrine cells (47). Basal cells are abundant in the lung and can also produce cytokines, including IL-33 (41).

Brush cells are not normally detected in healthy lungs, but a recent study demonstrated their presence following influenza virus infection (48). These chemosensory cells are analogous to the previously described Tuft cells in the gut, where a feed forward loop exists in which Tuft cell–derived IL-25 directs ILC2 expansion and ILC2-derived IL-13 promotes reciprocal Tuft cell expansion (49–51). Brush cells might therefore be an important source of IL-25 in the airway during allergic asthma, a notion supported by the finding that these cells are present at high numbers in polyps of individuals with chronic rhinosinusitis (52).

Pulmonary neuroendocrine cells (PNECs) are another relatively rare EC population found primarily near respiratory tree branch points. These cells are innervated and likely function to sense environmental changes and promote immune responses through production of neuropeptides (53). Intriguingly, PNECs colocalize with ILC2, suggesting these two cell types might communicate with one another. In support of this, PNEC production of calcitonin gene-related peptide (CGRP) enhances ILC2 production of IL-5 (54). In parallel, PNEC production of a different molecule, the neurotransmitter γ-aminobutyric acid (GABA), induces goblet cell hyperplasia. Lungs from human asthmatics show increased PNECs, suggesting that these cells are important contributors to allergic asthma (54).

Ionocytes are a third relatively rare population of recently discovered AECs (39). The name ionocyte reflects their transcription profile, which has striking similarities to specialized ion-transporting cells in the kidney. Of note, these cells also express high levels of the cystic fibrosis transmembrane receptor and likely contribute to mucus viscosity. Whether ionocytes also contribute more directly to immune responses in the lung remains to be determined.

The above-mentioned EC types in the lung represent the best-characterized ECs to date. However, recent scRNA-seq studies have revealed extensive heterogeneity in ECs in the human lung, with at least 10 distinct populations of ECs in the upper airways, lower airways, and parenchyma (55). A better understanding of this complexity and the functions each population fulfills is likely to reveal additional pathways that can be targeted for therapeutic gain.

A frequently used approach to study the relative impact of gene disruptions in structural cells and leukocytes is to generate reciprocal BM chimeric mice in which BM cells lacking a gene of interest are transplanted into wild-type mice and vice versa. ECs are relatively radio-resistant, and they are therefore often assumed to be the major cell type in the radio-resistant cell compartment that contributes to immune responses. Unfortunately, irradiation induces profound transcriptional changes in ECs that have unknown effects on their function (56). Moreover, large numbers of host macrophages and DCs remain in the lung after irradiation and this confounds interpretation of such studies (57). For these reasons, genetic engineering approaches to selectively delete genes in discrete cell types offers an attractive alternative for studying cell-specific gene function.

Reverse genetics, or the study of how various gene disruptions affect observable phenotypes, has had a powerful impact on biological studies over the last three decades. Cre recombinase–mediated genetic recombination has been, and continues to be, widely used to generate planned changes to the mouse genome. In the most widely used approach, the gene of interest (or a portion thereof) is flanked by Cre recognition sites (LoxP), and recombination across those sites deletes the intervening genomic DNA, thus ablating gene function. A wide variety of mice are now available in which Cre is expressed in a cell-specific manner. Some of these mouse strains have been used to study how expression of various genes by nonhematopoietic cells impacts immune responses in the lung.

The Vav1 gene is expressed in almost all hematopoietic cells, thus Vav-cre transgenes have been used to effectively express Cre in these cells (58). Comparisons of genes important in whole body knockout mice but not in Vav-cre–expressing mice are, therefore, useful in evaluating the involvement nonhematopoietic cells in the pathogenesis of allergic airway inflammation. However, some “off target” promoter expression occurs in nonhematopoietic cells (59).

Floxed genes of choice can be selectively deleted in virtually all pulmonary ECs using mice expressing Cre recombinase under the transcriptional control of the surfactant C promoter (Sftpc-cre) (60, 61). Several other promoters have been used to selectively express Cre in specific EC subsets, including the Secretoglobin1a1 (Scgb1a/CC10/CCSP) promoter to drive Cre in club cells, the Foxj1-cre promoter, which has been used for ciliated ECs (46), and Ager-creER for AT1 cells (62). Moreover, tamoxifen-inducible forms of Cre (CreER) impart temporal inducibility, as exemplified by the inducible and selective expression of Sftpc-creER transgenes in AT2 cells of adult mice (63).

Data from BM chimeras and Cre-mediated recombination have revealed that expression of Tlr4 in nonhematopoietic tissue-resident cells is required for eosinophilic airway inflammation; conversely, Tlr4 expression in hematopoietic cells promotes neutrophilic inflammation (64, 65). Similarly, manipulation of the adaptor protein Myd88 required for canonical TLR and IL-1R signaling revealed a similar, differential impact of nonhematopoietic and hematopoietic cell populations on eosinophilic and neutrophilic airway inflammation, respectively (61).

Transcriptomic signatures of conventional DCs in the lung are influenced by Myd88 signaling in ECs, suggesting that ECs may alert lung DCs to tissue damage or cellular stress, and that this might contribute to allergic sensitization (61). However, the molecular mechanisms that support allergic sensitization in experimental animal models depend on the adjuvant used. Thus, whereas TLR ligands (and allergens that contain endotoxin) induce IL-1α, IL-1β, TNF-α, and GM-CSF (66), this is not true for protease adjuvants (67). Nonetheless, the pathways stemming from TLR-dependent and protease-dependent allergic sensitization converge on EC-derived IL-33, as this cytokine is important in both of these general pathways (68, 69). Taken together, these findings show that innate immune signaling in ECs leads ultimately to eosinophilic inflammation of the airway and imply that EC → APC crosstalk influences allergic airway sensitization (Fig. 2B).

A wide array of EC-produced cytokines have been shown to act on immune cells, but much remains to be learned. Inhaled house dust extract triggers the release of endogenous, cell-free factors into the airway that license conventional DCs to promote Th2 differentiation (70). Although different features of lung DCs are activated by distinct pathways, it is of considerable interest to identify EC-derived factors contribute to allergic airway inflammation. It is likely that a variety of nonhematopoietic cell–derived molecules contribute to allergic responses in asthma through mechanisms involving 1) secreted soluble products, 2) extracellular vesicles (EVs), and 3) direct cell–cell interactions (Fig. 2C).

AECs secrete a wide variety of soluble products that can have powerful effects on both allergic sensitization and responses to subsequent allergen exposure, as summarized in Table I. A large number of chemokines contribute to the induction and maintenance of allergic asthma, as reviewed elsewhere (71, 72). These include CCL20, which recruits CCR6-expressing DCs and Th17 cells to the airway during sensitization, CXCL1 and CXCL5 that promote neutrophil recruitment, and CCL3 and CCL24 that drive airway eosinophilia. EC-derived purine nucleotides, such as ATP, can activate DCs during allergic sensitization (71) and also promote mast cell degranulation during the allergen challenge phase (73). Defensins are EC-derived peptides whose production increases in asthma. These peptides can act directly to kill bacteria and also promote immune responses, in part by binding to the chemokine receptor, CCR6, that is expressed on immature DCs (74). Surfactants not only prevent collapse of the alveoli during exhalation, but also have innate immune functions in the airways, including modulating LPS-, IL-13–, and aeroallergen-induced inflammation (75–77). The epithelium is also a rich source of innate immune cytokines. CSF-2 and CSF-1 can activate APCs, thereby promoting allergic sensitization and amplifying established inflammation, respectively. It is now well established that IL-25, thymic stromal lymphopoietin (TSLP), and IL-33 can drive allergic inflammation. Each of these cytokines act on ILC2s, but there are some differences in their actions (78). Thus, IL-33 is a particularly potent activator of ILC2s, whereas IL-25 can elicit production of IL-17 in addition to T2 cytokines (79). IL-25, IL-33, and TSLP can also activate other cells, including fibroblasts, ECs, endothelial cells, and circulating Th2 cells. These factors may also drive steroid-resistant asthma by way of ILC2 expansion.

The epithelium is also a major source of TGF-β, which is well known for its ability to suppress inflammation. Paradoxically, however, this cytokine is required for the generation and maintenance of ILC2 progenitors and for upregulation of the IL-33R IL-1RL1 (80). Consequently, selective deletion of TGF-β in AECs leads to fewer IL-13⁺ ILC2s and reduced inflammation in a chronic model of house dust mite–mediated allergic inflammation, despite no reduction in numbers of Th2 cells (81).

Although many cytokines are likely released unprotected, they would be subject to an array of degradative proteases and nucleases present in the bronchoalveolar space. Alternatively, cytokines and other mediators derived from nonhematopoietic cells could be carried from one cell to another within protective EVs. EVs can carry several types of cargo with relevance to asthma, including PGs (82), enzymes involved in the formation of leukotriene D4 (a potent mediator of bronchoconstriction) (83, 84), and microRNAs (miRNAs) that can modify the activity of other cells upon uptake (85, 86). Sequence analysis of miRNAs found in from EVs in the bronchoalveolar lavage fluid (BALF) show a close relationship to miRNAs found in ECs. In agreement with this, selective membrane-tagging experiments demonstrated that 80% of the EVs in BALF are derived from ECs, not hematopoietic cells (87). Comparatively little is known of the cytokine and enzyme content of EVs in the lung, but in the gut, EC-derived EVs have been shown to contain IL-1β and caspase 8 (88). Whether this also occurs in the lung to promote IL-1β–dependent immune responses, such as allergen-specific Th17 responses in steroid-resistant asthma, is currently unknown. It will be interesting to determine whether the contents of EC-derived miRNA-laden EVs in the lung are changed by inhalation of adjuvants and allergens and whether EVs can be taken up by APCs in the lung to modify responses to inhaled allergens.

During inflammation, a subset of alveolar macrophages (AMs) form connexin 43 (Cx43)–containing gap junctions with ECs. During LPS-induced inflammation, some AMs remain sessile and establish intercommunication through synchronized Ca(2+) waves, using the epithelium as the conducting pathway (89). The intercommunication involving Ca(2+)-dependent activation of Akt is immunosuppressive, as AM-specific knockout of Cx43 enhanced alveolar neutrophil recruitment and secretion of proinflammatory cytokines in the BALF. However, in COVID-19, disease severity correlates with stronger interactions between ECs and immune cells (90).

Inhalation of pollutants, pathogens, and allergens can lead to apoptosis of lung ECs, which can be cleared by macrophages or neighboring AECs through a process known as efferocytosis. EC-mediated efferocytosis is anti-inflammatory, as it increases production of IL-10 and reduces production of IL-33. It is also dependent on the GTPase Rac1, and deletion of this gene leads to increased inflammation characterized by increased IL-33 and decreased IL-10 (91). Moreover, macrophages can direct this process by releasing cytokines and microvesicles. This process is at least partly dependent on IGF-1, as deletion of its receptor on ECs leads to increased allergen-induced inflammation (92). Emerging evidence from apoptotic lymphocytes has revealed that during death, apoptotic cells release a specialized set of metabolites that induce specific gene programs in neighboring cells, including wound healing and suppression of inflammation (93). Whether AECs function similarly remains to be seen.

Primary ECs express Cd1d and other molecules involved in lipid Ag presentation (94). Additionally, AECs selectively express MHC class II (MHC-II). Evidence to date suggests that MHC-II–expressing ECs are tolerogenic in homeostasis. Nonetheless, MHC-II expression is demonstrably enhanced on AECs from patients with asthma and viral infection (95). The functional implication of Ag presentation by AECs necessitates further investigation to discern its impacts on the pathogenesis of various asthma endotypes.

Like ECs, other nonhematopoietic cells, including fibroblasts, vascular endothelial cells, follicular DCs, and nerves located within the lamina propria are also endowed with immunomodulatory capacities (96). The contributions of these latter cell populations to asthma remain incompletely understood. In particular, neuroimmune crosstalk is a burgeoning area of research with promising opportunities for therapeutic intervention for asthma (97). The lungs are supplied with a rich network of efferent and afferent nerves, and in addition to the production of the neuropeptides CGRP and GABA by innervated PNECs (53), neuronal modulation of airway smooth muscle tone, mucus secretion, and inflammation is an important factor in asthma pathogenesis. The underlying mechanism involves neuroimmune feedback in which nerves respond to inflammatory cues within the lung by releasing a diverse array of neuropeptides and other immunomodulatory factors (98). Evidence from experimental models suggests that nerves promote AHR and inflammation in asthma in part by activating ILC2s (99–101). IL-33 signaling in sensory nerves is a critical mediator of pruritus in contact dermatitis (102), but it remains to be determined whether neuronal IL-33 signaling likewise exacerbates inflammation in asthma.

Innate immune memory is another topic of growing interest. First identified in the skin, inflammatory memory is a relatively new concept that describes the ability of nonimmune cells, including ECs, to adapt to a stimulus and respond differently upon a second challenge (103). Recent multiorgan bioinformatics analysis revealed that nonhematopoietic structural cells exhibit epigenetic predilections for immune gene activation (31). Mechanistically, inflammatory memory can result from epigenetic modification of histones, a major mechanism for controlling gene expression in many settings, including asthma (104). For example, EC production of IL-17–induced neutrophil chemoattractants, such as CXCL1, is epigenetically controlled by a mechanism involving histone deacetylase (HDAC) 5 and bromodomain and extraterminal domain (BET) (105). Such epigenetic control might explain the ability of primary cells from individuals with chronic inflammation to spontaneously produce IL-17. Similarly, the observation that many allergic individuals develop sensitivity to additional allergens as they age, also known as the atopic march, might result from epigenetic reprogramming of proinflammatory genes in ECs and other nonhematopoietic cell populations. An improved mechanistic understanding of epigenetic reprograming might lead to new therapeutic opportunities.

Recent advances in scRNA-seq have also opened new frontiers into our understanding of how relatively rare cell populations affect the lung in health and disease (55, 106). Efforts are underway to consolidate scRNA-seq data onto platforms that are accessible to all investigators. These include two prominent platforms, known as the Tabula Muris (107), and the Human Lung Atlas (108). The latter is an offshoot from the larger Human Cell Atlas (www.humancellatlas.org/), which aims to characterize all cell types in the human body and how they change in disease. Moreover, Vieira Braga et al. (55) recently applied scRNA-seq to pulmonary cell subsets. In this study, patients with childhood-onset asthma displayed increased goblet cell numbers relative to healthy volunteers, and also had a novel ciliated cell type with ectopic expression of Muc5ac, termed “mucous ciliated cells.” Analysis of genes previously linked to asthma by GWAS studies revealed that many of these genes were highly expressed in ciliated cells and in the novel mucous ciliated cells (55). Another study also found transcriptional differences in ECs in polyps of patients with chronic rhinosinusitis (109). One limitation of scRNA-seq is the relatively shallow “depth” of sequencing for individual cells, which largely restricts discovery to highly expressed genes. Limitations in numbers of cells that are sequenced can preclude identification of rare cell types, such as brush cells and neuroendocrine cells (55). It is likely that this difficulty will be at least partially overcome as the technology continues to advance.

As more nonhematopoietic cell–expressed genes of interest are identified through RNA sequencing efforts, it will be necessary to test their function through reverse genetics. To this end, Cre-mediated recombination has been used to tremendous success, but expression of a single gene does not usually define a specific cell type. Accordingly, strategies have been developed to use multiple recombinases to achieve greater cell specificity (110). This approach was recently used to precisely target bronchioalveolar stem cells and follow their fate by lineage tracing (111). Another recent advance in reverse genetics is the use of clustered regularly interspersed short palindromic repeats (CRISPR)–mediated genome editing (112, 113). The main attraction of this approach is that requires only two elements: a bacterial Cas9 DNA nuclease, and a single-guide RNA (sgRNA) that directs this nuclease to a specified target site in the genome. CRISPR-mediated changes could include the introduction of LoxP sites to generate Cre-dependent, conditionally mutant mice. Use of this technology in vivo has been facilitated by the generation of transgenic mice expressing Cas9. In these animals, all that is needed for gene editing is the sgRNA, which can be provided through various means, including lentiviruses (114, 115). Drawbacks of the latter approach include a relatively inefficient transduction of lung ECs, and long-term effects of sgRNA expression, including off target effects and unwanted immune responses. As sgRNA delivery strategies advance, it should soon be possible to rapidly and efficiently target any gene of interest in the lung epithelium.

It is now clear that nonhematopoietic cells in the lung have many functions in addition to their well-established roles in gas exchange, barrier maintenance, and mucociliary clearance. These cells are exquisitely sensitive to both inhaled environmental agents and tissue damage, and responses to these challenges include the elaboration of both soluble and EV-enclosed factors that promote inflammation or, where appropriate, tissue repair. Tissue-resident nonhematopoietic cell responses are intimately intertwined with a variety of immune cells, and together, these two general cell types orchestrate responses that maintain pulmonary health. Advances in reverse genetics, cell lineage tracing, and scRNA-seq have provided and will continue to provide novel insights into the development and function of various nonhematopoietic cells in the lung. This new knowledge will in turn reveal new opportunities to harness nonhematopoietic cell function in targeted therapies designed to treat distinct forms of asthma.

The authors have no financial conflicts of interest.