Cellular crosstalk in the development and regeneration of the respiratory system

Abstract

The respiratory system, including the peripheral lungs, large airways and trachea, is one of the most recently evolved adaptations to terrestrial life. To support the exchange of respiratory gases, the respiratory system is interconnected with the cardiovascular system, and this interconnective nature requires a complex interplay between a myriad of cell types. Until recently, this complexity has hampered our understanding of how the respiratory system develops and responds to postnatal injury to maintain homeostasis. The advent of new single-cell sequencing technologies, developments in cellular and tissue imaging and advances in cell lineage tracing have begun to fill this gap. The view that emerges from these studies is that cellular and functional heterogeneity of the respiratory system is even greater than expected and also highly adaptive. In this Review, we explore the cellular crosstalk that coordinates the development and regeneration of the respiratory system. We discuss both the classic cell and developmental biology studies and recent single-cell analysis to provide an integrated understanding of the cellular niches that control how the respiratory system develops, interacts with the external environment and responds to injury.

Fig. 1 |. Cellular composition of airways.

a | The proximal region of the lower respiratory tract consists of the trachea, which proceeds distally to generate branching large and small airways, which terminate in alveoli. These airway tubes are wrapped by smooth muscle. b,c | Respiratory epithelium in the mouse and the human consists of many distinct cell types: goblet cells secrete mucus to entrap inhaled particulates; multiciliated cells expel the mucus together with the entrapped particulates; club cells are secretory cells that produce various factors with protective and immunomodulatory functions and also serve in detoxification of harmful substances; basal cells are progenitor cells for the airway epithelia; pulmonary neuroendocrine cells (PNECs; solitary as well as in clusters known as neuroendocrine bodies (NEBs)) probe the microenvironment to influence smooth muscle tone as well as regulate immune responses. There are also rare epithelial cell types, including brush (tuft) cells, which may serve an important role in regulating allergen driven type 2 immune responses, and the recently identified ionocytes, which appear to be a main source of cystic fibrosis transmembrane conductance regulator (CFTR) activity, thereby regulating mucus production (with a potential role in cystic fibrosis). Stromal cells such as airway smooth muscle (ASM), fibroblasts (sonic hedgehog responsive (Gli1 positive) and WNT responsive (Axin2 positive)) provide ligands and extracellular matrix that modulate airway epithelial cell turnover and restrict airway tube diameter. The tracheal region and large airways in the human respiratory tract and the trachea in mice harbour cartilaginous rings and submucosal glands. The latter contain mucous cells, serous cells and myoepithelial cells and, together with goblet cells, are responsible for the production of luminal mucus together with goblet cells (panel b). Unlike in the human, mouse small airways do not contain basal or goblet cells (panel c). Key markers expressed in each cell type shown in panels b and c are indicated.

Fig. 2 |. Cellular composition of alveoli.

a | The small airways terminate in alveolar sacs, where the inhaled air is circulated and oxygen-carbon dioxide gas exchange occurs within the capillary plexus. b | The alveolar niche is composed of alveolar type 1 (AT1) cells and alveolar type 2 (AT2) cells. AT1 cells are thin and elongated and cover the gas exchange surface area, remaining in close contact with the capillary plexus. AT2 cells produce pulmonary surfactant (stored in the lamellar bodies in the cytoplasm), which preserves surface tension of the alveolus to prevent collapse during breathing. Interstitial fibroblasts consisting of Axin2-positive myogenic precursors (AMPs), Wnt2-expressing platelet-derived growth factor-α (PDGFRa)-positive cells (WNT2-Pa) and mesenchymal alveolar niche cells (MANCs) make up most of the alveolar mesenchyme. These cells largely support alveolar structure by producing extracellular matrix as well as expressing ligands that support epithelial cell proliferation and differentiation (see also Fig. 5). Additionally, there are sentinel immune cells such as alveolar and interstitial macrophages that constantly survey the alveolar microenvironment for harmful pathogens or particulates. ASM, airway smooth muscle.

Fig. 3 |. Respiratory tract development and endoderm-mesoderm interactions.

a | By embryonic day 12.5 in mouse the NKX2.1-postive endoderm expresses SOX2 and SOX9 in proximal and distal cells, respectively. At this point branching morphogenesis has begun and the endoderm projects outward towards the FGF10-expressing mesoderm (dashed outline; see panel b). b | The respiratory mesoderm, which is derived from cardiopulmonary progenitors and expresses TBX4, is shown encapsulated by mesothelium. Mesodermal cells express FGF10 in the regions adjacent to the distal tip endoderm. Primitive airway smooth muscle (ASM), tracheal cartilage and the vascular tree are being specified during this time. c | The distal tip endoderm expresses SOX9 and ID2. These cells respond to FGF10 and WNTs derived from the mesoderm, which together stimulate their outgrowth. In a feedback response, the endoderm expresses ligands, such as sonic hedgehog (SHH) and WNT7b, that stimulate the differentiation of the mesoderm to ASM. d,e | Alveologenesis starts with the formation of primitive alveoli, which are circular and bulb-like (saccular stage; from embryonic day 17.5 to postnatal day 5 in the mouse). These primitive alveoli form at the termini of the bronchial tree and harbour flattened alveolar type 1 (AT1) cells as well as alveolar type 2 (AT2) cells, which remain cuboidal. During the alveolar stage (postnatal days 5 to 30 in the mouse) the formation of secondary septae occurs. This remodelling process increases the total surface area of the lung alveolus and is thought to be driven by mesenchymal cells, such as the secondary crest myofibroblasts (SCMFs). SCMFs are stimulated by epithelium-derived PDGFA and SHH. It is postulated that the contractile activity of the SCMFs physically shapes the alveolus. Additionally, WNT-responsive AT2 cells (AT2^Axin cells) proliferate during this time (receiving WNT ligands from mesenchymal cells) and increase production of pulmonary surfactant, which is critical for the transition to air breathing. Furthermore, the capillary plexus becomes more closely aligned with AT1 cells during alveologenesis.

Fig. 4 |. The airway niche turnover at homeostasis and in response to injury.

a | The turnover of the airway epithelium is low during homeostasis. Nevertheless, basal cells continuously self-renew at a low rate and preferentially differentiate towards the secretory cell lineage over the multiciliated cell lineage, which is regulated by Notch signalling within the basal cell population. Secretory cells have the capability to trans differentiate into multiciliated cells, which is inhibited by Notch signalling. The surrounding airway smooth muscle (ASM) tissue harbours LGR6-expressing cells that exhibit some homeostatic turnover. The epithelium secretes sonic hedgehog (SHH) to actively suppress the proliferation of peribronchiolar mesenchyme. b | Airway injury stimulates multiple cell types to regenerate the tissue. In response to tissue injury, basal cells are able to give rise to both secretory and multiciliated cells, likely reflecting the heterogeneity in the lineage (NOTCH2-expressing versus MYB-expressing subpopulations). Loss of SHH signalling between the epithelium and mesenchyme also relieves the block on mesenchymal cell proliferation. This loss of mesenchymal quiescence was shown to feedback on the epithelium and increase epithelial cell proliferation. In naphthalene injury models that mainly kill secretory cells (greyed-out cells) the spared or uninjured secretory cells (also referred to as ‘variant secretory cells’) proliferate and repopulate the lost secretory cells. Downregulation of Hippo signalling after airway injury causes YAP- mediated increase in epithelial WNT7b expression, which promotes FGF10 expression in the adjoining ASM. ASM-derived FGF10 then stimulates secretory cell proliferation and differentiation. Epithelial cell death induced by injury can also trigger a wound healing response that leads to the recruitment of inflammatory cells and subsequent expansion of mesenchymal cell populations.

Fig. 5 |. Response of the alveolar niche to tissue injury.

a | The alveolar niche has two types of epithelial cell: alveolar type 1 (AT1) cells and alveolar type 2 (AT2) cells. The latter includes a WNT-responsive AT2 subset of cells that have been shown to serve as alveolar epithelial progenitors. At homeostasis the alveolar epithelial progenitors self-renew and differentiate into AT1 cells. These processes are regulated by bone morphogenetic protein (BMP) signalling, which restricts AT2 self-renewal, favouring AT1 differentiation. The rates of this homeostatic cell turnover are generally low. The stromal population comprises platelet-derived growth factor receptor- a-expressing fibroblasts, such as mesenchymal alveolar niche cells (MANCs), which are situated next to AT2 cells and Axin2-positive myofibrogenic precursors (AMPs), which are sparsely located throughout the alveoli and near blood vessels. b | In response to tissue injury, the alveolar niche upregulates ligands that promote AT2 cell proliferation and differentiation into AT1 cells (which are predominantly lost in response to insults; indicated in grey). Pulmonary capillary endothelial cells (PCECs) have been shown to respond to injury by upregulating matrix metalloproteinase 14 (MMP14), which by digesting the extracellular matrix (ECM) releases epidermal growth factor (EGF)-like ligands that stimulate epithelial cell growth. Injury- activated MANCs promote AT2 proliferation and self-renewal by producing FGF7 and IL-6 as well as BMP antagonists such as NBL1, GREM2 and FSTL1. AT2 cell-produced chemokines and cytokines, including CCL2 and IL-33, recruit monocytes (which differentiate into macrophages) and innate lymphoid cells (ILCs). Innate lymphoid cells support epithelial regeneration in part by dampening the acute inflammatory responses and by modulating the polarization of macrophages to the alternative (M2) lineage, which promotes AT2 cell proliferation likely via mechanisms involving ECM remodelling. c | Severe or chronic lung injury can result in dysplastic responses — fibrosis (left) and generation of abnormal cell clusters (right) — that if left unchecked will ultimately impair lung compliance and gas exchange. The fibrotic response is derived from fibroblasts — mostly AMPs and to a lesser extent MANCs — that proliferate on injury and transition into collagen-producing, alpha smooth muscle actin (αSMA)-positive myofibroblasts that deposit excessive amounts of ECM, which severely impairs lung compliance. A crucial signal for this myofibrogenic transition is provided by transforming growth factor-β(TGFβ), which is derived from the surrounding ECM. In the setting of severe alveolar epithelial cell destruction, such as that triggered by influenza infection, distally located SOX2-positive and TRP63-positive basal cells migrate out of the distal medium and/or small airways to regions of injury in the alveoli and expand as KRT5-expressing epithelial-like pods that radiate out of the tissue. The formation of these pods responds to hypoxia and is thought to be an epithelial cell-derived wound healing response that maintains the tissue barrier but ultimately does not generate functional alveolar epithelium.