Lung megakaryocytes display distinct transcriptional and phenotypic properties.

Megakaryocytes (MKs) are responsible for platelet biogenesis, which is believed to occur canonically in adult bone marrow (BM) and in the fetal liver during development. However, emerging evidence highlights the lung as a previously underappreciated residence for MKs that may contribute significantly to circulating platelet mass. Although a diversity of cells specific to the BM is known to promote the maturation and trafficking of MKs, little investigation into the impact of the lung niche on the development and function of MKs has been done. Here, we describe the application of single-cell RNA sequencing, coupled with histological, ploidy, and flow cytometric analyses, to profile primary MKs derived from syngeneic mouse lung and hematopoietic tissues. Transcriptional profiling demonstrated that lung MKs have a unique signature distinct from their hematopoietic counterparts, with lung MKs displaying enrichment for maturation markers, potentially indicating a propensity for more efficient platelet production. Reciprocally, fetal lung MKs also showed the robust expression of cytokines and growth factors that are known to promote lung development. Lastly, lung MKs possess an enrichment profile skewed toward roles in immunity and inflammation. These findings highlight the existence of a lung-specific MK phenotype and support the notion that the lung plays an independent role in the development and functional maturation of MKs. The immune phenotype displayed by lung MKs also introduces their potential role in microbial surveillance and antigen presentation.

Airway-Associated Macrophages in Homeostasis and Repair.

There is an increasing appreciation for the heterogeneity of myeloid lineages in the lung, but relatively little is known about populations specifically associated with the conducting airways. We use single-cell RNA sequencing, flow cytometry, and immunofluorescence to characterize myeloid cells of the mouse trachea during homeostasis and epithelial injury/repair. We identify submucosal macrophages, similar to lung interstitial macrophages, and intraepithelial macrophages. Following injury, there are early increases in neutrophils and submucosal macrophages, including M2-like macrophages. Intraepithelial macrophages are lost after injury and later restored by CCR2+ monocytes. We show that repair of the tracheal epithelium is impaired in Ccr2-deficient mice. Mast cells and group 2 innate lymphoid cells are sources of interleukin-13 (IL-13) that polarize macrophages and directly influence basal cell behaviors. Their proximity to the airway epithelium establishes these myeloid populations as potential therapeutic targets for airway disease.

TMEM16A calcium-activated chloride currents in supporting cells of the mouse olfactory epithelium.

Glial-like supporting (or sustentacular) cells are important constituents of the olfactory epithelium that are involved in several physiological processes such as production of endocannabinoids, insulin, and ATP and regulation of the ionic composition of the mucus layer that covers the apical surface of the olfactory epithelium. Supporting cells express metabotropic P2Y purinergic receptors that generate ATP-induced Ca2+ signaling through the activation of a PLC-mediated cascade. Recently, we reported that a subpopulation of supporting cells expresses also the Ca2+-activated Cl- channel TMEM16A. Here, we sought to extend our understanding of a possible physiological role of this channel in the olfactory system by asking whether Ca2+ can activate Cl- currents mediated by TMEM16A. We use whole-cell patch-clamp analysis in slices of the olfactory epithelium to measure dose-response relations in the presence of various intracellular Ca2+ concentrations, ion selectivity, and blockage. We find that knockout of TMEM16A abolishes Ca2+-activated Cl- currents, demonstrating that TMEM16A is essential for these currents in supporting cells. Also, by using extracellular ATP as physiological stimuli, we found that the stimulation of purinergic receptors activates a large TMEM16A-dependent Cl- current, indicating a possible role of TMEM16A in ATP-mediated signaling. Altogether, our results establish that TMEM16A-mediated currents are functional in olfactory supporting cells and provide a foundation for future work investigating the precise physiological role of TMEM16A in the olfactory system.

Epithelial Chloride Transport by CFTR Requires TMEM16A.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is the secretory chloride/bicarbonate channel in airways and intestine that is activated through ATP binding and phosphorylation by protein kinase A, but fails to operate in cystic fibrosis (CF). TMEM16A (also known as anoctamin 1, ANO1) is thought to function as the Ca2+ activated secretory chloride channel independent of CFTR. Here we report that tissue specific knockout of the TMEM16A gene in mouse intestine and airways not only eliminates Ca2+-activated Cl- currents, but unexpectedly also abrogates CFTR-mediated Cl- secretion and completely abolishes cAMP-activated whole cell currents. The data demonstrate fundamentally new roles of TMEM16A in differentiated epithelial cells: TMEM16A provides a mechanism for enhanced ER Ca2+ store release, possibly engaging Store Operated cAMP Signaling (SOcAMPS) and activating Ca2+ regulated adenylyl cyclases. TMEM16A is shown to be essential for proper activation and membrane expression of CFTR. This intimate regulatory relationship is the cause for the functional overlap of CFTR and Ca2+-dependent chloride transport.

Recruited Monocytes and Type 2 Immunity Promote Lung Regeneration following Pneumonectomy.

To investigate the role of immune cells in lung regeneration, we used a unilateral pneumonectomy model that promotes the formation of new alveoli in the remaining lobes. Immunofluorescence and single-cell RNA sequencing found CD115+ and CCR2+ monocytes and M2-like macrophages accumulating in the lung during the peak of type 2 alveolar epithelial stem cell (AEC2) proliferation. Genetic loss of function in mice and adoptive transfer studies revealed that bone marrow-derived macrophages (BMDMs) traffic to the lung through a CCL2-CCR2 chemokine axis and are required for optimal lung regeneration, along with Il4ra-expressing leukocytes. Our data suggest that these cells modulate AEC2 proliferation and differentiation. Finally, we provide evidence that group 2 innate lymphoid cells are a source of IL-13, which promotes lung regeneration. Together, our data highlight the potential for immunomodulatory therapies to stimulate alveologenesis in adults.

Telomere dysfunction in alveolar epithelial cells causes lung remodeling and fibrosis.

Telomeres are short in type II alveolar epithelial cells (AECs) of patients with idiopathic pulmonary fibrosis (IPF). Whether dysfunctional telomeres contribute directly to development of lung fibrosis remains unknown. The objective of this study was to investigate whether telomere dysfunction in type II AECs, mediated by deletion of the telomere shelterin protein TRF1, leads to pulmonary fibrosis in mice (SPC-Cre TRF1fl/fl mice). Deletion of TRF1 in type II AECs for 2 weeks increased γH2AX DNA damage foci, but not histopathologic changes in the lung. Deletion of TRF1 in type II AECs for up to 9 months resulted in short telomeres and lung remodeling characterized by increased numbers of type II AECs, α-smooth muscle actin+ mesenchymal cells, collagen deposition, and accumulation of senescence-associated ß-galactosidase+ lung epithelial cells. Deletion of TRF1 in collagen-expressing cells caused pulmonary edema, but not fibrosis. These results demonstrate that prolonged telomere dysfunction in type II AECs, but not collagen-expressing cells, leads to age-dependent lung remodeling and fibrosis. We conclude that telomere dysfunction in type II AECs is sufficient to cause lung fibrosis, and may be a dominant molecular defect causing IPF. SPC-Cre TRF1fl/fl mice will be useful for assessing cellular and molecular mechanisms of lung fibrosis mediated by telomere dysfunction.

Lineage-negative progenitors mobilize to regenerate lung epithelium after major injury.

Broadly, tissue regeneration is achieved in two ways: by proliferation of common differentiated cells and/or by deployment of specialized stem/progenitor cells. Which of these pathways applies is both organ- and injury-specific. Current models in the lung posit that epithelial repair can be attributed to cells expressing mature lineage markers. By contrast, here we define the regenerative role of previously uncharacterized, rare lineage-negative epithelial stem/progenitor (LNEP) cells present within normal distal lung. Quiescent LNEPs activate a ΔNp63 (a p63 splice variant) and cytokeratin 5 remodelling program after influenza or bleomycin injury in mice. Activated cells proliferate and migrate widely to occupy heavily injured areas depleted of mature lineages, at which point they differentiate towards mature epithelium. Lineage tracing revealed scant contribution of pre-existing mature epithelial cells in such repair, whereas orthotopic transplantation of LNEPs, isolated by a definitive surface profile identified through single-cell sequencing, directly demonstrated the proliferative capacity and multipotency of this population. LNEPs require Notch signalling to activate the ΔNp63 and cytokeratin 5 program, and subsequent Notch blockade promotes an alveolar cell fate. Persistent Notch signalling after injury led to parenchymal 'micro-honeycombing' (alveolar cysts), indicative of failed regeneration. Lungs from patients with fibrosis show analogous honeycomb cysts with evidence of hyperactive Notch signalling. Our findings indicate that distinct stem/progenitor cell pools repopulate injured tissue depending on the extent of the injury, and the outcomes of regeneration or fibrosis may depend in part on the dynamics of LNEP Notch signalling.

Anoctamins support calcium-dependent chloride secretion by facilitating calcium signaling in adult mouse intestine.

Intestinal epithelial electrolyte secretion is activated by increase in intracellular cAMP or Ca(2+) and opening of apical Cl(-) channels. In infants and young animals, but not in adults, Ca(2+)-activated chloride channels may cause secretory diarrhea during rotavirus infection. While detailed knowledge exists concerning the contribution of cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) channels, analysis of the role of Ca(2+)-dependent Cl(-) channels became possible through identification of the anoctamin (TMEM16) family of proteins. We demonstrate expression of several anoctamin paralogues in mouse small and large intestines. Using intestinal-specific mouse knockout models for anoctamin 1 (Ano1) and anoctamin 10 (Ano10) and a conventional knockout model for anoctamin 6 (Ano6), we demonstrate the role of anoctamins for Ca(2+)-dependent Cl(-) secretion induced by the muscarinic agonist carbachol (CCH). Ano1 is preferentially expressed in the ileum and large intestine, where it supports Ca(2+)-activated Cl(-) secretion. In contrast, Ano10 is essential for Ca(2+)-dependent Cl(-) secretion in jejunum, where expression of Ano1 was not detected. Although broadly expressed, Ano6 has no role in intestinal cholinergic Cl(-) secretion. Ano1 is located in a basolateral compartment/membrane rather than in the apical membrane, where it supports CCH-induced Ca(2+) increase, while the essential and possibly only apical Cl(-) channel is CFTR. These results define a new role of Ano1 for intestinal Ca(2+)-dependent Cl(-) secretion and demonstrate for the first time a contribution of Ano10 to intestinal transport.

Sizing up lung stem cells.

Mammalian lungs are comprised of conducting airways and alveoli. How the distinct epithelial linings of these two zones are differentially specified and maintained is not fully understood. In this issue of Developmental Cell, two groups find critical roles for the Hippo pathway in regulation of lung progenitor cell differentiation.

The calcium-activated chloride channel Anoctamin 1 contributes to the regulation of renal function.

The role of calcium-activated chloride channels for renal function is unknown. By immunohistochemistry we demonstrate dominant expression of the recently identified calcium-activated chloride channels, Anoctamin 1 (Ano1, TMEM16A) in human and mouse proximal tubular epithelial (PTE) cells, with some expression in podocytes and other tubular segments. Ano1-null mice had proteinuria and numerous large reabsorption vesicles in PTE cells. Selective knockout of Ano1 in podocytes (Ano1-/-/Nphs2-Cre) did not impair renal function, whereas tubular knockout in Ano1-/-/Ksp-Cre mice increased urine protein excretion and decreased urine electrolyte concentrations. Purinergic stimulation activated calcium-dependent chloride currents in isolated proximal tubule epithelial cells from wild-type but not from Ano1-/-/Ksp-Cre mice. Ano1 currents were activated by acidic pH, suggesting parallel stimulation of Ano1 chloride secretion with activation of the proton-ATPase. Lack of calcium-dependent chloride secretion in cells from Ano1-/-/Ksp-Cre mice was paralleled by attenuated proton secretion and reduced endosomal acidification, which compromised proximal tubular albumin uptake. Tubular knockout of Ano1 enhanced serum renin and aldosterone concentrations, probably leading to enhanced compensatory distal tubular reabsorption, thus maintaining normal blood pressure levels. Thus, Ano1 has a role in proximal tubular proton secretion and protein reabsorption. The results correspond to regulation of the proton-ATPase by the Ano1-homolog Ist2 in yeast.

Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction.

Mucous cell hyperplasia and airway smooth muscle (ASM) hyperresponsiveness are hallmark features of inflammatory airway diseases, including asthma. Here, we show that the recently identified calcium-activated chloride channel (CaCC) TMEM16A is expressed in the adult airway surface epithelium and ASM. The epithelial expression is increased in asthmatics, particularly in secretory cells. Based on this and the proposed functions of CaCC, we hypothesized that TMEM16A inhibitors would negatively regulate both epithelial mucin secretion and ASM contraction. We used a high-throughput screen to identify small-molecule blockers of TMEM16A-CaCC channels. We show that inhibition of TMEM16A-CaCC significantly impairs mucus secretion in primary human airway surface epithelial cells. Furthermore, inhibition of TMEM16A-CaCC significantly reduces mouse and human ASM contraction in response to cholinergic agonists. TMEM16A-CaCC blockers, including those identified here, may positively impact multiple causes of asthma symptoms.

TMEM16A induces MAPK and contributes directly to tumorigenesis and cancer progression.

Frequent gene amplification of the receptor-activated calcium-dependent chloride channel TMEM16A (TAOS2 or ANO1) has been reported in several malignancies. However, its involvement in human tumorigenesis has not been previously studied. Here, we show a functional role for TMEM16A in tumor growth. We found TMEM16A overexpression in 80% of head and neck squamous cell carcinoma (SCCHN), which correlated with decreased overall survival in patients with SCCHN. TMEM16A overexpression significantly promoted anchorage-independent growth in vitro, and loss of TMEM16A resulted in inhibition of tumor growth both in vitro and in vivo. Mechanistically, TMEM16A-induced cancer cell proliferation and tumor growth were accompanied by an increase in extracellular signal-regulated kinase (ERK)1/2 activation and cyclin D1 induction. Pharmacologic inhibition of MEK/ERK and genetic inactivation of ERK1/2 (using siRNA and dominant-negative constructs) abrogated the growth effect of TMEM16A, indicating a role for mitogen-activated protein kinase (MAPK) activation in TMEM16A-mediated proliferation. In addition, a developmental small-molecule inhibitor of TMEM16A, T16A-inh01 (A01), abrogated tumor cell proliferation in vitro. Together, our findings provide a mechanistic analysis of the tumorigenic properties of TMEM16A, which represents a potentially novel therapeutic target. The development of small-molecule inhibitors against TMEM16A may be clinically relevant for treatment of human cancers, including SCCHN.

Evidence for type II cells as cells of origin of K-Ras-induced distal lung adenocarcinoma.

Identifying the cells of origin of lung cancer may lead to new therapeutic strategies. Previous work has focused upon the putative bronchoalveolar stem cell at the bronchioalveolar duct junction as a cancer cell of origin when a codon 12 K-Ras mutant is induced via adenoviral Cre inhalation. In the present study, we use two "knock-in" Cre-estrogen receptor alleles to inducibly express K-RasG12D in CC10(+) epithelial cells and Sftpc(+) type II alveolar cells of the adult mouse lung. Analysis of these mice identifies type II cells, Clara cells in the terminal bronchioles, and putative bronchoalveolar stem cells as cells of origin for K-Ras-induced lung hyperplasia. However, only type II cells appear to progress to adenocarcinoma.

Ano1 as a regulator of proliferation.

Ano1 is a recently discovered Ca(2+)-activated Cl(-) channel expressed on interstitial cells of Cajal (ICC) that has been implicated in slow-wave activity in the gut. However, Ano1 is expressed on all classes of ICC, even those that do not contribute to generation of the slow wave, suggesting that Ano1 may have an alternate function in these cells. Ano1 is also highly expressed in gastrointestinal stromal tumors. Mice lacking Ano1 had fewer proliferating ICC in whole mount preparations and in culture, raising the possibility that Ano1 is involved in proliferation. Cl(-) channel blockers decreased proliferation in cells expressing Ano1, including primary cultures of ICC and in the pancreatic cancer-derived cell line, CFPAC-1. Cl(-) channel blockers had a reduced effect on Ano1(-/-) cultures, confirming that the blockers are acting on Ano1. Ki67 immunoreactivity, 5-ethynyl-2'-deoxyuridine incorporation, and cell-cycle analysis of cells grown in low-Cl(-) media showed fewer proliferating cells than in cultures grown in regular medium. We confirmed that mice lacking Ano1 had less phosphorylated retinoblastoma protein compared with controls. These data led us to conclude that Ano1 regulates proliferation at the G(1)/S transition of the cell cycle and may play a role in tumorigenesis.

Epithelial progenitor cells in lung development, maintenance, repair, and disease.

The vertebrate lung is elegantly patterned to carry out gas exchange and host defense. Similar to other organ systems, endogenous stem and progenitor cells fuel the organogenesis of the lung and maintain homeostasis in the face of normal wear and tear. In the context of acute injury, these progenitor populations are capable of effecting efficient repair. However, chronic injury, inflammation, and immune rejection frequently result in pathological airway remodeling and serious impairment of lung function. Here, we review the development, maintenance, and repair of the vertebrate respiratory system with an emphasis on the roles of epithelial stem and progenitor cells. We discuss what is currently known about their identities, lineage relationships, and the mechanisms that regulate their differentiation along various lineages. A deeper understanding of these progenitor populations will undoubtedly accelerate the discovery of improved cellular, genetic, molecular, and bioengineered therapies for lung disease.

Notch-dependent differentiation of adult airway basal stem cells.

The epithelium lining the airways of the adult human lung is composed of ciliated and secretory cells together with undifferentiated basal cells (BCs). The composition and organization of this epithelium is severely disrupted in many respiratory diseases. However, little is known about the mechanisms controlling airway homeostasis and repair after epithelial damage. Here, we exploit the mouse tracheobronchial epithelium, in which BCs function as resident stem cells, as a genetically tractable model of human small airways. Using a reporter allele we show that the low level of Notch signaling at steady state is greatly enhanced during repair and the generation of luminal progenitors. Loss-of-function experiments show that Notch signaling is required for the differentiation, but not self-renewal, of BCs. Moreover, sustained Notch activation in BCs promotes their luminal differentiation, primarily toward secretory lineages. We also provide evidence that this function of Notch signaling is conserved in BCs from human airways.

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

The small airways of the human lung undergo pathological changes in pulmonary disorders, such as chronic obstructive pulmonary disease (COPD), asthma, bronchiolitis obliterans and cystic fibrosis. These clinical problems impose huge personal and societal healthcare burdens. The changes, termed 'pathological airway remodeling', affect the epithelium, the underlying mesenchyme and the reciprocal trophic interactions that occur between these tissues. Most of the normal human airway is lined by a pseudostratified epithelium of ciliated cells, secretory cells and 6-30% basal cells, the proportion of which varies along the proximal-distal axis. Epithelial abnormalities range from hypoplasia (failure to differentiate) to basal- and goblet-cell hyperplasia, squamous- and goblet-cell metaplasia, dysplasia and malignant transformation. Mesenchymal alterations include thickening of the basal lamina, smooth muscle hyperplasia, fibrosis and inflammatory cell accumulation. Paradoxically, given the prevalence and importance of airway remodeling in lung disease, its etiology is poorly understood. This is due, in part, to a lack of basic knowledge of the mechanisms that regulate the differentiation, maintenance and repair of the airway epithelium. Specifically, little is known about the proliferation and differentiation of basal cells, a multipotent stem cell population of the pseudostratified airway epithelium. This Perspective summarizes what we know, and what we need to know, about airway basal cells to evaluate their contributions to normal and abnormal airway remodeling. We contend that exploiting well-described model systems using both human airway epithelial cells and the pseudostratified epithelium of the genetically tractable mouse trachea will enable crucial discoveries regarding the pathogenesis of airway disease.

Evidence that SOX2 overexpression is oncogenic in the lung.

BACKGROUND: SOX2 (Sry-box 2) is required to maintain a variety of stem cells, is overexpressed in some solid tumors, and is expressed in epithelial cells of the lung. METHODOLOGY/PRINCIPAL FINDINGS: We show that SOX2 is overexpressed in human squamous cell lung tumors and some adenocarcinomas. We have generated mouse models in which Sox2 is upregulated in epithelial cells of the lung during development and in the adult. In both cases, overexpression leads to extensive hyperplasia. In the terminal bronchioles, a trachea-like pseudostratified epithelium develops with p63-positive cells underlying columnar cells. Over 12-34 weeks, about half of the mice expressing the highest levels of Sox2 develop carcinoma. These tumors resemble adenocarcinoma but express the squamous marker, Trp63 (p63). CONCLUSIONS: These findings demonstrate that Sox2 overexpression both induces a proximal phenotype in the distal airways/alveoli and leads to cancer.

Loss of TMEM16A causes a defect in epithelial Ca2+-dependent chloride transport.

Molecular identification of the Ca(2+)-dependent chloride channel TMEM16A (ANO1) provided a fundamental step in understanding Ca(2+)-dependent Cl(-) secretion in epithelia. TMEM16A is an intrinsic constituent of Ca(2+)-dependent Cl(-) channels in cultured epithelia and may control salivary output, but its physiological role in native epithelial tissues remains largely obscure. Here, we demonstrate that Cl(-) secretion in native epithelia activated by Ca(2+)-dependent agonists is missing in mice lacking expression of TMEM16A. Ca(2+)-dependent Cl(-) transport was missing or largely reduced in isolated tracheal and colonic epithelia, as well as hepatocytes and acinar cells from pancreatic and submandibular glands of TMEM16A(-/-) animals. Measurement of particle transport on the surface of tracheas ex vivo indicated largely reduced mucociliary clearance in TMEM16A(-/-) mice. These results clearly demonstrate the broad physiological role of TMEM16A(-/-) for Ca(2+)-dependent Cl(-) secretion and provide the basis for novel treatments in cystic fibrosis, infectious diarrhea, and Sjöegren syndrome.

Expression of anoctamin 1/TMEM16A by interstitial cells of Cajal is fundamental for slow wave activity in gastrointestinal muscles.

Interstitial cells of Cajal (ICC) generate pacemaker activity (slow waves) in gastrointestinal (GI) smooth muscles, but the mechanism(s) of pacemaker activity are controversial. Several conductances, such as Ca(2+)-activated Cl() channels (CaCC) and non-selective cation channels (NSCC) have been suggested to be involved in slow wave depolarization. We investigated the expression and function of a new class of CaCC, anoctamin 1 (ANO1), encoded by Tmem16a, which was discovered to be highly expressed in ICC in a microarray screen. GI muscles express splice variants of the Tmem16a transcript in addition to other paralogues of the Tmem16a family. ANO1 protein is expressed abundantly and specifically in ICC in all regions of the murine, non-human primate (Macaca fascicularis) and human GI tracts. CaCC blocking drugs, niflumic acid and 4,4-diisothiocyano-2,2-stillbene-disulfonic acid (DIDS) reduced the frequency and blocked slow waves in murine, primate, human small intestine and stomach in a concentration-dependent manner. Unitary potentials, small stochastic membrane depolarizations thought to underlie slow waves, were insensitive to CaCC blockers. Slow waves failed to develop by birth in mice homozygous for a null allele of Tmem16a (Tmem16a(tm1Bdh)(/tm1Bdh)) and did not develop subsequent to birth in organ culture, as in wildtype and heterozygous muscles. Loss of function of ANO1 did not inhibit the development of ICC networks that appeared structurally normal as indicated by Kit antibodies. These data demonstrate the fundamental role of ANO1 in the generation of slow waves in GI ICC.