Spontaneous Activity of Cochlear Hair Cells Triggered by Fluid Secretion Mechanism in Adjacent Support Cells.

Spontaneous electrical activity of neurons in developing sensory systems promotes their maturation and proper connectivity. In the auditory system, spontaneous activity of cochlear inner hair cells (IHCs) is initiated by the release of ATP from glia-like inner supporting cells (ISCs), facilitating maturation of central pathways before hearing onset. Here, we find that ATP stimulates purinergic autoreceptors in ISCs, triggering Cl(-) efflux and osmotic cell shrinkage by opening TMEM16A Ca(2+)-activated Cl(-) channels. Release of Cl(-) from ISCs also forces K(+) efflux, causing transient depolarization of IHCs near ATP release sites. Genetic deletion of TMEM16A markedly reduces the spontaneous activity of IHCs and spiral ganglion neurons in the developing cochlea and prevents ATP-dependent shrinkage of supporting cells. These results indicate that supporting cells in the developing cochlea have adapted a pathway used for fluid secretion in other organs to induce periodic excitation of hair cells.

Human distal lung maps and lineage hierarchies reveal a bipotent progenitor.

Mapping the spatial distribution and molecular identity of constituent cells is essential for understanding tissue dynamics in health and disease. We lack a comprehensive map of human distal airways, including the terminal and respiratory bronchioles (TRBs), which are implicated in respiratory diseases1-4. Here, using spatial transcriptomics and single-cell profiling of microdissected distal airways, we identify molecularly distinct TRB cell types that have not-to our knowledge-been previously characterized. These include airway-associated LGR5+ fibroblasts and TRB-specific alveolar type-0 (AT0) cells and TRB secretory cells (TRB-SCs). Connectome maps and organoid-based co-cultures reveal that LGR5+ fibroblasts form a signalling hub in the airway niche. AT0 cells and TRB-SCs are conserved in primates and emerge dynamically during human lung development. Using a non-human primate model of lung injury, together with human organoids and tissue specimens, we show that alveolar type-2 cells in regenerating lungs transiently acquire an AT0 state from which they can differentiate into either alveolar type-1 cells or TRB-SCs. This differentiation programme is distinct from that identified in the mouse lung5-7. Our study also reveals mechanisms that drive the differentiation of the bipotent AT0 cell state into normal or pathological states. In sum, our findings revise human lung cell maps and lineage trajectories, and implicate an epithelial transitional state in primate lung regeneration and disease.

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.

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.

Differential sensitivity of gastric and small intestinal muscles to inducible knockdown of anoctamin 1 and the effects on gastrointestinal motility.

KEY POINTS: Electrical pacemaking in gastrointestinal muscles is generated by specialized interstitial cells of Cajal that produce the patterns of contractions required for peristalsis and segmentation in the gut. The calcium-activated chloride conductance anoctamin-1 (Ano1) has been shown to be responsible for the generation of pacemaker activity in GI muscles, but this conclusion is established from studies of juvenile animals in which effects of reduced Ano1 on gastric emptying and motor patterns could not be evaluated. Knocking down Ano1 expression using Cre/LoxP technology caused dramatic changes in in gastric motor activity, with disrupted slow waves, abnormal phasic contractions and delayed gastric emptying; modest changes were noted in the small intestine. Comparison of the effects of Ano1 antagonists on muscles from juvenile and adult small intestinal muscles suggests that conductances in addition to Ano1 may develop with age and contribute to pacemaker activity. ABSTRACT: Interstitial cells of Cajal (ICC) generate slow waves and transduce neurotransmitter signals in the gastrointestinal (GI) tract, facilitating normal motility patterns. ICC express a Ca2+ -activated Cl- conductance (CaCC), and constitutive knockout of the channel protein anoctamin-1 leads to loss of slow waves in gastric and intestinal muscles. These knockout experiments were performed on juvenile mice. However, additional experiments demonstrated significant differences in the sensitivity of gastric and intestinal muscles to antagonists of anoctamin-1 channels. Furthermore, the significance of anoctamin-1 and the electrical and mechanical behaviours facilitated by this conductance have not been evaluated on the motor behaviours of adult animals. Cre/loxP technology was used to generate cell-specific knockdowns of anoctamin-1 in ICC (KitCreERT2/+ ;Ano1tm2jrr/+ ) in GI muscles. The recombination efficiency of KitCreERT was evaluated with an eGFP reporter, molecular techniques and immunohistochemistry. Electrical and contractile experiments were used to examine the consequences of anoctamin-1 knockdown on pacemaker activity, mechanical responses, gastric motility patterns, gastric emptying and GI transit. Reduced anoctamin-1 caused loss of gastric, but not intestinal slow waves. Irregular spike complexes developed in gastric muscles, leading to uncoordinated antral contractions, delayed gastric emptying and increased total GI transit time. Slow waves in intestinal muscles of juvenile mice were more sensitive to anoctamin-1 antagonists than slow waves in adult muscles. The low susceptibility to anoctamin-1 knockdown and weak efficacy of anoctamin-1 antagonists in inhibiting slow waves in adult small intestinal muscles suggest that a conductance in addition to anoctamin-1 may develop in small intestinal ICC with ageing and contribute to pacemaker activity.

The cells and conductance mediating cholinergic neurotransmission in the murine proximal stomach.

KEY POINTS: Enteric neurotransmission is essential for gastrointestinal (GI) motility, although the cells and conductances responsible for post-junctional responses are controversial. The calcium-activated chloride conductance (CaCC), anoctamin-1 (Ano1), was expressed by intramuscular interstitial cells of Cajal (ICC-IM) in proximal stomach and not resolved in smooth muscle cells (SMCs). Cholinergic nerve fibres were closely apposed to ICC-IM. Conductances activated by cholinergic stimulation in isolated ICC-IM and SMCs were determined. A CaCC was activated by carbachol in ICC-IM and a non-selective cation conductance in SMCs. Responses to cholinergic nerve stimulation were studied. Excitatory junction potentials (EJPs) and mechanical responses were evoked in wild-type mice but absent or greatly reduced with knockout/down of Ano1. Drugs that block Ano1 inhibited the conductance activated by carbachol in ICC-IM and EJPs and mechanical responses in tissues. The data of the present study suggest that electrical and mechanical responses to cholinergic nerve stimulation are mediated by Ano1 expressed in ICC-IM and not SMCs. ABSTRACT: Enteric motor neurotransmission is essential for normal gastrointestinal (GI) motility. Controversy exists regarding the cells and ionic conductance(s) that mediate post-junctional neuroeffector responses to motor neurotransmitters. Isolated intramuscular ICC (ICC-IM) and smooth muscle cells (SMCs) from murine fundus muscles were used to determine the conductances activated by carbachol (CCh) in each cell type. The calcium-activated chloride conductance (CaCC), anoctamin-1 (Ano1) is expressed by ICC-IM but not resolved in SMCs, and CCh activated a Cl- conductance in ICC-IM and a non-selective cation conductance in SMCs. We also studied responses to nerve stimulation using electrical-field stimulation (EFS) of intact fundus muscles from wild-type and Ano1 knockout mice. EFS activated excitatory junction potentials (EJPs) in wild-type mice, although EJPs were absent in mice with congenital deactivation of Ano1 and greatly reduced in animals in which the CaCC-Ano1 was knocked down using Cre/loxP technology. Contractions to cholinergic nerve stimulation were also greatly reduced in Ano1 knockouts. SMCs cells also have receptors and ion channels activated by muscarinic agonists. Blocking acetylcholine esterase with neostigmine revealed a slow depolarization that developed after EJPs in wild-type mice. This depolarization was still apparent in mice with genetic deactivation of Ano1. Pharmacological blockers of Ano1 also inhibited EJPs and contractile responses to muscarinic stimulation in fundus muscles. The data of the present study are consistent with the hypothesis that ACh released from motor nerves binds muscarinic receptors on ICC-IM with preference and activates Ano1. If metabolism of acetylcholine is inhibited, ACh overflows and binds to extrajunctional receptors on SMCs, eliciting a slower depolarization response.

Etiology of distinct membrane excitability in pre- and posthearing auditory neurons relies on activity of Cl- channel TMEM16A.

The developmental rehearsal for the debut of hearing is marked by massive changes in the membrane properties of hair cells (HCs) and spiral ganglion neurons (SGNs). Whereas the underlying mechanisms for the developing HC transition to mature stage are understood in detail, the maturation of SGNs from hyperexcitable prehearing to quiescent posthearing neurons with broad dynamic range is unknown. Here, we demonstrated using pharmacological approaches, caged-Ca(2+) photolysis, and gramicidin patch recordings that the prehearing SGN uses Ca(2+)-activated Cl(-) conductance to depolarize the resting membrane potential and to prime the neurons in a hyperexcitable state. Immunostaining of the cochlea preparation revealed the identity and expression of the Ca(2+)-activated Cl(-) channel transmembrane member 16A (TMEM16A) in SGNs. Moreover, null deletion of TMEM16A reduced the Ca(2+)-activated Cl(-) currents and action potential firing in SGNs. To determine whether Cl(-) ions and TMEM16A are involved in the transition between pre- and posthearing features of SGNs we measured the intracellular Cl(-) concentration [Cl(-)]i in SGNs. Surprisingly, [Cl(-)]i in SGNs from prehearing mice was ∼90 mM, which was significantly higher than posthearing neurons, ∼20 mM, demonstrating discernible altered roles of Cl(-) channels in the developing neuron. The switch in [Cl(-)]i stems from delayed expression of the development of intracellular Cl(-) regulating mechanisms. Because the Cl(-) channel is the only active ion-selective conductance with a reversal potential that lies within the dynamic range of SGN action potentials, developmental alteration of [Cl(-)]i, and hence the equilibrium potential for Cl(-) (ECl), transforms pre- to posthearing phenotype.

Distinct stem cells contribute to mammary gland development and maintenance.

The mammary epithelium is composed of several cell lineages including luminal, alveolar and myoepithelial cells. Transplantation studies have suggested that the mammary epithelium is maintained by the presence of multipotent mammary stem cells. To define the cellular hierarchy of the mammary gland during physiological conditions, we performed genetic lineage-tracing experiments and clonal analysis of the mouse mammary gland during development, adulthood and pregnancy. We found that in postnatal unperturbed mammary gland, both luminal and myoepithelial lineages contain long-lived unipotent stem cells that display extensive renewing capacities, as demonstrated by their ability to clonally expand during morphogenesis and adult life as well as undergo massive expansion during several cycles of pregnancy. The demonstration that the mammary gland contains different types of long-lived stem cells has profound implications for our understanding of mammary gland physiology and will be instrumental in unravelling the cells at the origin of breast cancers.

Molecular Characterization of the Genital Organizer: Gene Expression Profile of the Mouse Urethral Plate Epithelium.

PURPOSE: Lower urinary tract malformations are among the most common congenital anomalies in humans. Molecular genetic studies of mouse external genital development have begun to identify mechanisms that pattern the genital tubercle and orchestrate urethral tubulogenesis. The urethral plate epithelium is an endodermal signaling region that has an essential role in external genital development. However, little is known about the molecular identity of this cell population or the genes that regulate its activity. MATERIALS AND METHODS: We used microarray analysis to characterize differences in gene expression between urethral plate epithelium and surrounding tissue in mouse genital tubercles. In situ hybridizations were performed to map gene expression patterns and ToppCluster (https://toppcluster.cchmc.org/) was used to analyze gene associations. RESULTS: A total of 84 genes were enriched at least 20-fold in urethral plate epithelium relative to surrounding tissue. The majority of these genes were expressed throughout the urethral plate in males and females at embryonic day 12.5 when the urethral plate is known to signal. Functional analysis using ToppCluster revealed genetic pathways with known functions in other organ systems but unknown roles in external genital development. Additionally, a 3-dimensional molecular atlas of genes enriched in urethral plate epithelium was generated and deposited at the GUDMAP (GenitoUrinary Development Molecular Anatomy Project) website (http://gudmap.org/). CONCLUSIONS: We identified dozens of genes previously unknown to be expressed in urethral plate epithelium at a crucial developmental period. It provides a novel panel of genes for analysis in animal models and in humans with external genital anomalies.

An Lmx1b-miR135a2 regulatory circuit modulates Wnt1/Wnt signaling and determines the size of the midbrain dopaminergic progenitor pool.

MicroRNAs regulate gene expression in diverse physiological scenarios. Their role in the control of morphogen related signaling pathways has been less studied, particularly in the context of embryonic Central Nervous System (CNS) development. Here, we uncover a role for microRNAs in limiting the spatiotemporal range of morphogen expression and function. Wnt1 is a key morphogen in the embryonic midbrain, and directs proliferation, survival, patterning and neurogenesis. We reveal an autoregulatory negative feedback loop between the transcription factor Lmx1b and a newly characterized microRNA, miR135a2, which modulates the extent of Wnt1/Wnt signaling and the size of the dopamine progenitor domain. Conditional gain of function studies reveal that Lmx1b promotes Wnt1/Wnt signaling, and thereby increases midbrain size and dopamine progenitor allocation. Conditional removal of Lmx1b has the opposite effect, in that expansion of the dopamine progenitor domain is severely compromised. Next, we provide evidence that microRNAs are involved in restricting dopamine progenitor allocation. Conditional loss of Dicer1 in embryonic stem cells (ESCs) results in expanded Lmx1a/b+ progenitors. In contrast, forced elevation of miR135a2 during an early window in vivo phenocopies the Lmx1b conditional knockout. When En1::Cre, but not Shh::Cre or Nes::Cre, is used for recombination, the expansion of Lmx1a/b+ progenitors is selectively reduced. Bioinformatics and luciferase assay data suggests that miR135a2 targets Lmx1b and many genes in the Wnt signaling pathway, including Ccnd1, Gsk3b, and Tcf7l2. Consistent with this, we demonstrate that this mutant displays reductions in the size of the Lmx1b/Wnt1 domain and range of canonical Wnt signaling. We posit that microRNA modulation of the Lmx1b/Wnt axis in the early midbrain/isthmus could determine midbrain size and allocation of dopamine progenitors. Since canonical Wnt activity has recently been recognized as a key ingredient for programming ESCs towards a dopaminergic fate in vitro, these studies could impact the rational design of such protocols.

Evidence for lung epithelial stem cell niches.

Recent studies have identified epithelial stem and progenitor cell populations of the lung. We are just beginning to understand the mechanisms that regulate their homeostatic, regenerative and maladaptive behaviors. Here, we discuss evidence of regulatory niches for epithelial stem cells of the lung.

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.

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.

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.

Neurotransmitter map of the asymmetric dorsal habenular nuclei of zebrafish.

The role of the habenular nuclei in modulating fear and reward pathways has sparked a renewed interest in this conserved forebrain region. The bilaterally paired habenular nuclei, each consisting of a medial/dorsal and lateral/ventral nucleus, can be further divided into discrete subdomains whose neuronal populations, precise connectivity, and specific functions are not well understood. An added complexity is that the left and right habenulae show pronounced morphological differences in many non-mammalian species. Notably, the dorsal habenulae of larval zebrafish provide a vertebrate genetic model to probe the development and functional significance of brain asymmetry. Previous reports have described a number of genes that are expressed in the zebrafish habenulae, either in bilaterally symmetric patterns or more extensively on one side of the brain than the other. The goal of our study was to generate a comprehensive map of the zebrafish dorsal habenular nuclei, by delineating the relationship between gene expression domains, comparing the extent of left-right asymmetry at larval and adult stages, and identifying potentially functional subnuclear regions as defined by neurotransmitter phenotype. Although many aspects of habenular organization appear conserved with rodents, the zebrafish habenulae also possess unique properties that may underlie lateralization of their functions.

Ano1, a Ca2+-activated Cl- channel, coordinates contractility in mouse intestine by Ca2+ transient coordination between interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) are pacemaker cells that generate electrical activity to drive contractility in the gastrointestinal tract via ion channels. Ano1 (Tmem16a), a Ca(2+)-activated Cl(-) channel, is an ion channel expressed in ICC. Genetic deletion of Ano1 in mice resulted in loss of slow waves in smooth muscle of small intestine. In this study, we show that Ano1 is required to maintain coordinated Ca(2+) transients between myenteric ICC (ICC-MY) of small intestine. First, we found spontaneous Ca(2+) transients in ICC-MY in both Ano1 WT and knockout (KO) mice. However, Ca(2+) transients within the ICC-MY network in Ano1 KO mice were uncoordinated, while ICC-MY Ca(2+) transients in Ano1 WT mice were rhythmic and coordinated. To confirm the role of Ano1 in the loss of Ca(2+) transient coordination, we used pharmacological inhibitors of Ano1 activity and shRNA-mediated knock down of Ano1 expression in organotypic cultures of Ano1 WT small intestine. Coordinated Ca(2+) transients became uncoordinated using both these approaches, supporting the conclusion that Ano1 is required to maintain coordination/rhythmicity of Ca(2+) transients. We next determined the effect on smooth muscle contractility using spatiotemporal maps of contractile activity in Ano1 KO and WT tissues. Significantly decreased contractility that appeared to be non-rhythmic and uncoordinated was observed in Ano1 KO jejunum. In conclusion, Ano1 has a previously unidentified role in the regulation of coordinated gastrointestinal smooth muscle function through coordination of Ca(2+) transients in ICC-MY.

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.

Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition.

There are currently few treatment options for pulmonary fibrosis. Innovations may come from a better understanding of the cellular origin of the characteristic fibrotic lesions. We have analyzed normal and fibrotic mouse and human lungs by confocal microscopy to define stromal cell populations with respect to several commonly used markers. In both species, we observed unexpected heterogeneity of stromal cells. These include numerous cells with molecular and morphological characteristics of pericytes, implicated as a source of myofibroblasts in other fibrotic tissues. We used mouse genetic tools to follow the fates of specific cell types in the bleomcyin-induced model of pulmonary fibrosis. Using inducible transgenic alleles to lineage trace pericyte-like cells in the alveolar interstitium, we show that this population proliferates in fibrotic regions. However, neither these cells nor their descendants express high levels of the myofibroblast marker alpha smooth muscle actin (Acta2, aSMA). We then used a Surfactant protein C-CreER(T2) knock-in allele to follow the fate of Type II alveolar cells (AEC2) in vivo. We find no evidence at the cellular or molecular level for epithelial to mesenchymal transition of labeled cells into myofibroblasts. Rather, bleomycin accelerates the previously reported conversion of AEC2 into AEC1 cells. Similarly, epithelial cells labeled with our Scgb1a1-CreER allele do not give rise to fibroblasts but generate both AEC2 and AEC1 cells in response to bleomycin-induced lung injury. Taken together, our results show a previously unappreciated heterogeneity of cell types proliferating in fibrotic lesions and exclude pericytes and two epithelial cell populations as the origin of myofibroblasts.

A specialized population of monocyte-derived tracheal macrophages promote airway epithelial regeneration through a CCR2-dependent mechanism.

Macrophages are critical for maintenance and repair of mucosal tissues. While functionally distinct subtypes of macrophage are known to have important roles in injury response and repair in the lungs, little is known about macrophages in the proximal conducting airways. Single-cell RNA sequencing and flow cytometry demonstrated murine tracheal macrophages are largely monocyte-derived and are phenotypically distinct from lung macrophages at homeostasis. Following sterile airway injury, monocyte-derived macrophages are recruited to the trachea and activate a pro-regenerative phenotype associated with wound healing. Animals lacking the chemokine receptor CCR2 have reduced numbers of circulating monocytes and tracheal macrophages, deficient pro-regenerative macrophage activation and defective epithelial repair. Together, these studies indicate that recruitment and activation of monocyte-derived tracheal macrophages is CCR2-dependent and is required for normal airway epithelial regeneration.

IL-4-induced SOX9 confers lineage plasticity to aged adult lung stem cells.

Wound healing in response to acute injury is mediated by the coordinated and transient activation of parenchymal, stromal, and immune cells that resolves to homeostasis. Environmental, genetic, and epigenetic factors associated with inflammation and aging can lead to persistent activation of the microenvironment and fibrosis. Here, we identify opposing roles of interleukin-4 (IL-4) cytokine signaling in interstitial macrophages and type II alveolar epithelial cells (ATIIs). We show that IL4Ra signaling in macrophages promotes regeneration of the alveolar epithelium after bleomycin-induced lung injury. Using organoids and mouse models, we show that IL-4 directly acts on a subset of ATIIs to induce the expression of the transcription factor SOX9 and reprograms them toward a progenitor-like state with both airway and alveolar lineage potential. In the contexts of aging and bleomycin-induced lung injury, this leads to aberrant epithelial cell differentiation and bronchiolization, consistent with cellular and histological changes observed in interstitial lung disease.