Neuroepithelial Bodies Research Articles

Neuroepithelial bodies and terminal bronchioles are niches for distinctive club cells that repair the airways following acute notch inhibition

Lower airway club cells (CCs) serve the dual roles of a secretory cell and a stem cell. Here, we probe how theCC fate is regulated. We find that, in response to acute perturbation of Notch signaling, CCs adopt distinct fates. Although the vast majority transdifferentiate into multiciliated cells, a "variant" subpopulation (v-CCs), juxtaposed to neuroepithelial bodies (NEBs; 5%-10%) and located at bronchioalveolar duct junctions (>80%), does not. Instead, v-CCs transition into lineage-ambiguous states but can revert to a CC fate upon restoration of Notch signaling and repopulate the airways with CCs and multiciliated cells. The v-CC response to Notch inhibition is dependent on localized activation of β-catenin in v-CCs. We propose that the CC fate is stabilized by canonical Notch signaling, that airways are susceptible to perturbations to this pathway, and that NEBs/terminal bronchioles comprise niches that modulate CC plasticity via β-catenin activation to facilitate airway repair post Notch inhibition.

GABA-immunoreactivity in Neuroepithelial Bodies of Spontaneously Hypertensive Rats

Neuroepithelial bodies (NEBs) are complex sensory receptors dispersed throughout the intrapulmonary airways. They are composed of pulmonary neuroendocrine cells (PNECs) which have a large amount of dense-core vesicles containing calcitonin gene-related peptide (CGRP), serotonin (5HT), substance P (SP) and δ-aminobutyric acid (GABA). NEBs are densely innervated by vagal sensory terminals, dorsal root afferents and intrinsic motor fibres. Their location, neurochemical composition and dense innervation make NEBs a key component for the regulation of lung homeostasis. Pulmonary arterial hypertension is a rare disease characterized by morphological alterations in the microcirculation of the lungs causing a wide array of complications and high mortality. Spontaneously hypertensive rats (SHRs) are a common model for essential hypertension, that may develop secondary pulmonary hypertension and thus can be used as an animal model to study pulmonary pressure changes. The present study focuses on the immunohistochemical demonstration of GABA in the NEBs and its potential role in the regulation of the pulmonary pressure. Two groups of SHRs were used and age-matched normotensive Wistar Kyoto (WKY) rats served as controls. We found single PNECs and clusters of GABA-immunoreactive cells protruding into the lumen of the intrapulmonary airways in all examined groups of SHRs and in the normotensive WKY rats. However, the expressional levels statistically differed within the two age groups of SHRs and the controls. Specifically, the NEBs and PNECs in 1-month-old SHRs and particularly in 3-month-old SHRs showed more intense GABA-immunostaining compared to the WKY rats. In conclusion, NEBs and PNECs react to the alterations of the pulmonary pressure homeostasis by an increased GABA expression as a preventive or reactive regulation mechanism against developing pulmonary hypertension.

The pulmonary neuroepithelial body-vagal sensory nerve axis is altered in a mouse model of bronchopulmonary dysplasia (BPD)

Breathing control problems, like apnea of prematurity, are a common complication in preterm infants with bronchopulmonary dysplasia (BPD), but the underlying factors remain incompletely understood. A subpopulation of pulmonary vagal afferent nerve fibers (Calb1+) innervate CO2 and O2 sensitive clusters of lung neuroendocrine cells (neuroepithelial bodies (NEBs)) located along conducting airways. Calb1+ fibers provide direct sensory input to the brainstem that putatively limit tidal volume. NEBs are hyperplastic in BPD infants and may increase activity of inhibitory Calb1+ vagal fibers. Herein, we sought to determine if the NEB-Calb1 lung-brainstem axis is altered in association with breathing control in BPD. BPD in mice was established by exposure to chronic neonatal (postnatal day (P) 0-14) hyperoxia (Hx; 70% O2; n=20) or normoxia (Nx; n=17). Consistent with human BPD, Hx caused alveolar simplification supported by a reduction in alveolar density and alveoli (p<0.05; separate t-tests) in P22 H&E-stained lung sections (n=5-7/group). Like human infants at risk for BPD, Hx mice adapted to increased dead space (caused by reduction in alveoli) by hyperventilating (p<0.05; generalized linear modeling accounting for fat free mass and heat production, measured by NMR and respirometry; n=10-24/group). This was facilitated by an increase in tidal volume that was significantly reduced at P14 but increased at P17 and P21 (n=19-28) (Tukey’s post-hoc: p<0.05). NEBs and associated innervation (vagal and dorsal root ganglia) were next assessed in subsets of mice. First, IT instillation of WGA-594 lectin into subsets of mice (n=9 mice/18 ganglia per group) and subsequent quantification of 594+ neurons per vagal ganglia revealed fewer neurons (p=0.07; t-test) in Hx mice (110.8 ± 6.9 vs 95.2 ± 4.9). Second, CGRP+ innervation to NEBs is reduced in Hx mice (Quantification of CGRP+ nerve fibers only: 17740.4 +/- 4126 vs 10474.6 +/- 2140 +CGRP pixels; p=0.12 n=5-7/group). Third, there was greater frequency of NEBs per unit airway (p<0.05; 0.306 ± 0.03 vs 0.401 ± 0.03) with a tendency for larger NEBs (p=0.06; 9.14 ± 0.7 vs 11.93 ± 1.3) indicating NEB hyperplasia. Together, these data indicate individual NEB innervation is reduced in BPD but that total NEB innervation may remain unchanged. If this is true for Calb1+ vagal fibers remain untested. To functionally assess Calb1-vagal fibers we activated, with DCZ, the excitatory synthetic Gq-DREAD receptor (DTR) expressed in Calb1cre mice via microinjection of AAV-Cre-Gq-mCherry into the left vagal ganglia. DCZ but not vehicle caused a major increase in peak inspiratory flow during acute hypoxic challenges compared to vehicle treated mice (Pre-DMSO, Post-DMSO, Pre-DCZ = ~1ml/sec vs 4ml/sec post-DCZ; p<0.05, n=4/group). Collection of vagal ganglia confirms DTR expression but spillover of DTR is present. Repeating these studies with diluted AAV-Cre-Gq in control and BPD mice are ongoing. Together, our data verify that hyperoxia induced BPD in mice causes a similar reduction in alveoli and compensation to increased dead space by hyperventilation as observed in human BPD. Additionally, BPD structurally alters the NEB-vagal axis but whether these changes are functionally significant to nCOB is being addressed with ongoing studies. AHA 20CDA35310121, Advancing Healthier Wisconsin Endowment. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A vagal reflex evoked by airway closure

Airway integrity must be continuously maintained throughout life. Sensory neurons guard against airway obstruction and, on a moment-by-moment basis, enact vital reflexes to maintain respiratory function1,2. Decreased lung capacity is common and life-threatening across many respiratory diseases, and lung collapse can be acutely evoked by chest wall trauma, pneumothorax or airway compression. Here we characterize a neuronal reflex of the vagus nerve evoked by airway closure that leads to gasping. In vivo vagal ganglion imaging revealed dedicated sensory neurons that detect airway compression but not airway stretch. Vagal neurons expressing PVALB mediate airway closure responses and innervate clusters of lung epithelial cells called neuroepithelial bodies (NEBs). Stimulating NEBs or vagal PVALB neurons evoked gasping in the absence of airway threats, whereas ablating NEBs or vagal PVALB neurons eliminated gasping in response to airway closure. Single-cell RNA sequencing revealed that NEBs uniformly express the mechanoreceptor PIEZO2, and targeted knockout of Piezo2 in NEBs eliminated responses to airway closure. NEBs were dispensable for the Hering–Breuer inspiratory reflex, which indicated that discrete terminal structures detect airway closure and inflation. Similar to the involvement of Merkel cells in touch sensation3,4, NEBs are PIEZO2-expressing epithelial cells and, moreover, are crucial for an aspect of lung mechanosensation. These findings expand our understanding of neuronal diversity in the airways and reveal a dedicated vagal pathway that detects airway closure to help preserve respiratory function.

THU121 Dopamine Regulates Neuroendocrine Cell Expansion In The Developing Lung

Abstract Disclosure: T. Watanabe: None. M. Stupnikov: None. Y. Zhou: None. X. Ke: None. J. Qien: None. A. Ozawa: None. M. Horigome: None. M. Yamada: None. M. Mori: None. W.V. Cardoso: None. Pulmonary neuroendocrine cells (PNECs) are rare airway epithelial cells in the lung that have function as neurosensory and endocrine cells. They arise from naive epithelial progenitor cells in mice on embryonic day (E) 12.5, migrate to branch point of proximal airway and rare found as solitary or in clusters of 5 to 20 cells termed neuroepithelial bodies (NEBs). PNECs can be aberrantly increased in a variety of human diseases, including asthma, neuroendocrine hyperplasia of infants (NEHI) and sudden infant death syndrome (SIDS). Ascl1, an Achaete-Scute family bHLH transcriptional factor is the earliest marker and master regulator of PNEC fate specification. No PNECs form in Ascl1 null mice. In an effort to identify Ascl1 downstream targets involved in PNEC specification and expansion, we performed transcriptional analysis of E14.5 lungs from Ascl1 null mouse. Gene Ontology analysis showed a number of genes associated with the catecholamine pathway downregulated in mutants, compared to WT. These included tyrosine hydroxylase (Th) dopa decarboxylase (Ddc), dopamine beta-hydroxylase (Dβh). To gain initial insights into how catecholamine signaling influence PNEC development, we cultured E12.5 lung explants with various inhibitors of the catecholamine biosynthesis pathway and examined the effects on cell differentiation and proliferation. Treatment with Metyrosine (Met) and Benserazide (Benz) which inhibits dopamine production, resulted in a striking increase in PNEC number in NEBs as seen by expansion in Ascl1+ cells, without affecting cell proliferation. By contrast, no effect was observed with Zamicastat (Zami) which inhibits norepinephrine production. To examine whether dopamine regulated PNEC differentiation, we used the D1-like receptor antagonist, SCH-23390 and the D2-like receptor antagonist, Haloperidol in lung explant cultures, as before. Only Haloperidol increased PNECs in cultured lungs. This effect was similarly observed by maternal administration of Haloperidol intraperitoneally from E9.5 to 13.5 and analysis of harvested embryos at E14.5 and 18.5. Current studies mapping expression of other components of the dopamine pathway in the developing airway epithelium suggests a complex interaction of PNECs with neighbor cells in the regulation of the proper number of NEBs and solitary NE cells. These observations may provide a link between catecholamine signaling and aberrant PNEC behavior in the pathogenesis of human conditions affecting these cells. Presentation: Thursday, June 15, 2023

The NEB-P2ry1 lung-brainstem axis and the control of breathing in a mouse model of bronchopulmonary dysplasia

Breathing control problems, like apnea of prematurity, are a common complication in preterm infants with bronchopulmonary dysplasia (BPD), but the underlying factors remain incompletely understood. A subpopulation of pulmonary vagal afferent nerve fibers (P2ry1+) innervate CO2 and O2 sensitive clusters of lung neuroendocrine cells (neuroepithelial bodies (NEBs)) located along conducting airways. P2ry1 fibers provide direct sensory input to the brainstem and inhibit breathing upon stimulation. NEBs are hyperplastic in BPD infants and may increase activity of inhibitory P2ry1 vagal fibers. Herein, we sought to determine if the NEB-P2ry1 lung-brainstem axis is altered in association with breathing control in BPD. BPD in mice was induced by exposure to chronic neonatal (postnatal day (P) 0-14) hyperoxia (Hx; 70% O2; n=20) or normoxia (Nx; n=17). Consistent with human BPD, Hx caused alveolar simplification supported by a reduction in alveolar density (p<0.05) and alveoli (p<0.05) in P22 H&E-stained lung sections. Hx mice spent more time apneic at P14 and P17 (p<0.05) in room air and acute hypoxia. Overall (main effect), room air minute ventilation (VE) was reduced in HX mice (p<0.05). Further, VE was reduced during acute hypoxia at P14 and P17 (not P21) with an overall reduction of breathing in Hx mice (p<0.05). From a subset of mice (n=4-5/group) VCO2, VO2, and heat production at rest were greater in P21 Hx mice during room air, suggesting the Hx mice were hypoventilating. Immunofluorescent staining of CGRP (hallmark NEB marker), Ki67 (proliferation), and SEZ6L2 (new NEB cell-membrane marker) in Hx and Nx lungs were imaged and analyzed by a blinded investigator. Consistent with clinical findings, Hx mice had hyperplastic NEBs (8.6 ± 0.45 vs 10.7 ± 1.3 cells/NEB; p<0.05) while only 2 of 143 NEBs expressed Ki67, suggesting no proliferation at P22. CGRP co-localized with SEZ6L2 in both Nx and Hx lungs. A subset of mice received intratracheal delivery of 1ug SEZ6L2-Saporin toxin to lesion NEBs and assess their function. 4 days after SEZ-Saporin treatment breathing frequency was elevated by 11% ± 5% in room air and by 7% ± 5% during acute hypoxia but not in PBS-treated, adult mice (n=3/group) suggesting NEBs have a mild inhibitory influence under normal conditions. Lastly, wireless optogenetic stimulation of P2ry1 vagal fibers in unanesthetized adult control mice (n=3) increased room air breathing by 20% ± 1% and drive to breathe by 36% ± 19% compared to pre-stim breathing. Together, our data demonstrate that hyperoxia-induced BPD alters the control of breathing which may in part be mediated via a unique lung-brainstem axis comprised of hyperplastic NEBs causing greater inhibition of excitatory P2ry1 vagal fibers. The NEB-P2ry1 axis may serve as a novel target for improving breathing in BPD. GCM: Advancing Healthier Wisconsin REAC Award, AHA 20CDA35310121; MRH: HL122358; JLG: DK133121, HL134850, and HL084207; JLS: DK133121 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The pulmonary neuroepithelial body microenvironment represents an underestimated multimodal component in airway sensory pathways.

Exciting new imaging and molecular tools, combined with state-of-the-art genetically modified mouse models, have recently boosted interest in pulmonary (vagal) sensory pathway investigations. In addition to the identification of diverse sensory neuronal subtypes, visualization of intrapulmonary projection patterns attracted renewed attention on morphologically identified sensory receptor end-organs, such as the pulmonary neuroepithelial bodies (NEBs) that have been our area of expertise for the past four decades. The current review aims at providing an overview of the cellular and neuronal components of the pulmonary NEB microenvironment (NEB ME) in mice, underpinning the role of these complexly organized structures in the mechano- and chemosensory potential of airways and lungs. Interestingly, the pulmonary NEB ME additionally harbors different types of stem cells, and emerging evidence suggests that the signal transduction pathways that are active in the NEB ME during lung development and repair also determine the origin of small cell lung carcinoma. Although documented for many years that NEBs appear to be affected in several pulmonary diseases, the current intriguing knowledge on the NEB ME seems to encourage researchers that are new to the field to explore the possibility that these versatile sensor-effector units may be involved in lung pathogenesis or pathobiology.

Microscopic Anatomy of the Pulmonary Neuroepithelial Bodies in Spontaneously Hypertensive Rats

Microscopic Anatomy of the Pulmonary Neuroepithelial Bodies in Spontaneously Hypertensive Rats

Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies.

The use of organoids has become increasingly popular recently due to their self-organizing abilities, which facilitate developmental and disease modeling. Various methods have been described to create embryoid bodies (EBs) generated from embryonic or pluripotent stem cells but with varying levels of differentiation success and producing organoids of variable size. Commercial ultra-low attachment (ULA) V-bottom well plates are frequently used to generate EBs. These plates are relatively expensive and not as widely available as standard concave well plates. Here, we describe a cost-effective and low labor-intensive method that creates homogeneous EBs at high yield in standard V- and U-bottom well plates by applying an anti-adherence solution to reduce surface attachment, followed by centrifugation to enhance cellular aggregation. We also explore the effect of different seeding densities, in the range of 1 to 11 ×103 cells per well, for the fabrication of neuroepithelial EBs. Our results show that the use of V-bottom well plates briefly treated with anti-adherent solution (for 5 min at room temperature) consistently yields functional neural EBs in the range of seeding densities from 5 to 11×103 cells per well. A brief post-seeding centrifugation step further enhances EB establishment. EBs fabricated using centrifugation exhibited lower variability in their final size than their non-centrifuged counterparts, and centrifugation also improved EB yield. The span of conditions for reliable EB production is narrower in U-bottom wells than in V-bottom wells (i.e., seeding densities between 7×103 and 11×103 and using a centrifugation step). We show that EBs generated by the protocols introduced here successfully developed into neural organoids and expressed the relevant markers associated with their lineages. We anticipate that the cost-effective and easily implemented protocols presented here will greatly facilitate the generation of EBs, thereby further democratizing the worldwide ability to conduct organoid-based research.

Neonatal ventilatory changes in the control of breathing in a hyperoxia‐induced mouse model of bronchopulmonary dysplasia

While hyperoxia is needed for survival of very premature infants (<28 weeks gestation) due to immature lungs and immature neural control of breathing, it contributes to bronchopulmonary dysplasia (BPD), a chronic lung disease associated with heighted pediatric morbidity and mortality. Given the plasticity of the neural control of breathing, this study seeks to address whether the neural control of breathing adapts to lung disease and the roles of clusters of pulmonary neuroendocrine cells called neuroepithelial bodies (NEBs) in mediating breathing adaptations (or maladaptations). NEBs line the airways, are O2 and CO2 sensitive, are hypertrophied/hyperplastic in human BPD, and are innervated by vagal fibers that inhibit breathing when activated. Herein, we test the hypotheses that chronic neonatal hyperoxia causes age‐dependent changes to cardiorespiratory control and these changes are in part mediated by NEBs. Mice expressing eGFP in all NEBs were exposed to hyperoxia (FIO2: 0.7 from 0‐14 days of life; P0‐14) had reduced alveolar density (P<0.001) and fewer alveoli (P<0.001) compared with normoxic controls (n=4) at P22 (by 2‐tailed t‐Test), consistent with clinical BPD. Whole‐body flow through plethysmography did not reveal baseline breathing differences between normoxic (n=10) and hyperoxic (n=10) mice at any of the ages studied (P14‐22), though there is a trend (n=3/group) for a greater respiratory exchange ratio (RER) measured by respirometry in hyperoxic mice. There was also a significant main effect of hyperoxia (n=7) reducing the hypoxic and hypercapnic ventilatory responses compared to normoxic mice (n=10; two‐way mixed effect model with Sidak post‐hoc test). Ongoing studies aim to lesion NEBs using Saporin toxin conjugated to an NEB specific membrane protein in a subset of mice to address the functional role of NEBs in breathing in normoxic and hyperoxic treated mice.

Localizing the expression of Piezo2 in healthy and diseased lung tissue

Intractable air hunger remains one of the most impactful symptoms of chronic cardiopulmonary disease and current pharmacological management remains limited. However, an endogenous mechanism for air hunger relief exists. Pulmonary stretch receptors (PSRs) responding to lung inflation reduce the brainstem drive to breathe and mitigate air hunger. Harnessing this mechanism could form the basis of a novel therapy. Recent animal studies suggest Piezo2, a mechanically‐gated cation channel, causes PSR depolarization when airway smooth muscle is stretched during inflation. We have assessed whether Piezo2 is associated with PSRs in healthy and diseased human airways to determine whether Piezo2 is a potential pharmaceutical target.MethodsHuman lung biopsies from NIH Lung Tissue Research Consortium were used for identification of Piezo2 receptors in emphysema, fibrosis, bronchiolitis, and normal lung tissue. Localization of Piezo2 was determined with immunofluorescent staining and imaged with confocal microscopy (Zeiss LSM880). As PSR fiber endings are associated with airway smooth muscle, co‐staining for Smooth Muscle Antibody (SMA) and Calcitonin Gene‐Related Peptide (CGRP) was performed to determine whether Piezo2 is associated with PSR.Summary of resultsPiezo2 is associated with airway smooth muscle (as evidenced by SMA, figure 1) and neuroepithelial bodies in normal human lung tissue (evidenced by cGRP, figure 2) and expression persisted in all disease states tested.ConclusionsPiezo2 location is consistent with that of PSR in human lungs, making it a candidate for the mechanoreceptive transducer of human PSRs as has been demonstrated in animal studies. Expression persists in disease, making Piezo2 a potential therapeutic target, but it is still yet to be determined if Piezo2 is more significantly associated with airway or vascular smooth muscle in different disease states.

Mapping of the Sensory Innervation of the Mouse Lung by Specific Vagal and Dorsal Root Ganglion Neuronal Subsets.

The airways are densely innervated by sensory afferent nerves, whose activation regulates respiration and triggers defensive reflexes (e.g., cough, bronchospasm). Airway innervation is heterogeneous, and distinct afferent subsets have distinct functional responses. However, little is known of the innervation patterns of subsets within the lung. A neuroanatomical map is critical for understanding afferent activation under physiological and pathophysiological conditions. Here, we quantified the innervation of the mouse lung by vagal and dorsal root ganglion (DRG) sensory subsets defined by the expression of Pirt (all afferents), 5HT3 (vagal nodose afferents), Tac1 (tachykinergic afferents), and transient receptor potential vanilloid 1 channel (TRPV1; defensive/nociceptive afferents) using Cre-mediated reporter expression. We found that vagal afferents innervate almost all conducting airways and project into the alveolar region, whereas DRG afferents only innervate large airways. Of the two vagal ganglia, only nodose afferents project into the alveolar region, but both nodose and jugular afferents innervate conducting airways throughout the lung. Many afferents that project into the alveolar region express TRPV1. Few DRG afferents expressed TRPV1. Approximately 25% of blood vessels were innervated by vagal afferents (many were Tac1+). Approximately 10% of blood vessels had DRG afferents (some were Tac1+), but this was restricted to large vessels. Lastly, innervation of neuroepithelial bodies (NEBs) correlated with the cell number within the bodies. In conclusion, functionally distinct sensory subsets have distinct innervation patterns within the conducting airways, alveoli and blood vessels. Physiologic (e.g., stretch) and pathophysiological (e.g., inflammation, edema) stimuli likely vary throughout these regions. Our data provide a neuroanatomical basis for understanding afferent responses in vivo.

Mechanosensitivity of Murine Lung Slowly Adapting Receptors: Minimal Impact of Chemosensory, Serotonergic, and Purinergic Signaling.

Murine slowly adapting receptors (SARs) within airway smooth muscle provide volume-related feedback; however, their mechanosensitivity and morphology are incompletely characterized. We explored two aspects of SAR physiology: their inherent static mechanosensitivity and a potential link to pulmonary neuroepithelial bodies (NEBs). SAR mechanosensitivity displays a rate sensitivity linked to speed of inflation; however, to what extent static SAR mechanosensitivity is tuned for the very rapid breathing frequency (Bf) of small mammals (e.g., mouse) is unclear. NEB-associated, morphologically described smooth muscle-associated receptors (SMARs) may be a structural analog for functionally characterized SARs, suggesting functional linkages between SARs and NEBs. We addressed the hypotheses that: (1) rapid murine Bf is associated with enhanced in vivo SAR static sensitivity; (2) if SARs and NEBs are functionally linked, stimuli reported to impact NEB function would alter SAR mechanosensitivity. We measured SAR action potential discharge frequency (AP f, action potentials/s) during quasi-static inflation [0–20 cmH2O trans-respiratory pressure (PTR)] in NEB-relevant conditions of hypoxia (FIO2 = 0.1), hypercarbia (FICO2 = 0.1), and pharmacologic intervention (serotonergic 5-HT3 receptor antagonist, Tropisetron, 4.5 mg/kg; P2 purinergic receptor antagonist, Suramin, 50 mg/kg). In all protocols, we obtained: (1) AP f vs. PTR; (2) PTR threshold; and (3) AP f onset at PTR threshold. The murine AP f vs. PTR response comprises high AP f (average maximum AP f: 236.1 ± 11.1 AP/s at 20 cmH2O), a low PTR threshold (mean 2.0 ± 0.1 cmH2O), and a plateau in AP f between 15 and 20 cmH2O. Murine SAR mechanosensitivity (AP f vs. PTR) is up to 60% greater than that reported for larger mammals. Even the maximum difference between intervention and control conditions was minimally impacted by NEB-related alterations: Tropisetron −7.6 ± 1.8% (p = 0.005); Suramin −10.6 ± 1.5% (p = 0.01); hypoxia +9.3 ± 1.9% (p < 0.001); and hypercarbia −6.2 ± 0.9% (p < 0.001). We conclude that the high sensitivity of murine SARs to inflation provides enhanced resolution of operating lung volume, which is aligned with the rapid Bf of the mouse. We found minimal evidence supporting a functional link between SARs and NEBs and speculate that the <10% change in SAR mechanosensitivity during altered NEB-related stimuli is not consistent with a meaningful physiologic role.

CXCL13 is expressed in a subpopulation of neuroendocrine cells in the murine trachea and lung

The conducting airways are lined by distinct cell types, comprising basal, secretory, ciliated, and rare cells, including ionocytes, solitary cholinergic chemosensory cells, and solitary and clustered (neuroepithelial bodies) neuroendocrine cells. Airway neuroendocrine cells are in clinical focus since they can give rise to small cell lung cancer. They have been implicated in diverse functions including mechanosensation, chemosensation, and regeneration, and were recently identified as regulators of type 2 immune responses via the release of the neuropeptide calcitonin gene-related peptide (CGRP). We here assessed the expression of the chemokine CXCL13 (B cell attracting chemokine) by these cells by RT-PCR, in silico analysis of publicly available sequencing data sets, immunohistochemistry, and immuno-electron microscopy. We identify a phenotype of neuroendocrine cells in the naïve mouse, producing the chemokine CXCL13 predominantly in solitary neuroendocrine cells of the tracheal epithelium (approx. 70% CXCL13+) and, to a lesser extent, in the solitary neuroendocrine cells and neuroepithelial bodies of the intrapulmonary bronchial epithelium (< 10% CXCL13+). In silico analysis of published sequencing data of murine tracheal epithelial cells was consistent with the results obtained by immunohistochemistry as it revealed that neuroendocrine cells are the major source of Cxcl13-mRNA, which was expressed by 68–79% of neuroendocrine cells. An unbiased scRNA-seq data analysis of overall gene expression did not yield subclusters of neuroendocrine cells. Our observation demonstrates phenotypic heterogeneity of airway neuroendocrine cells and points towards a putative immunoregulatory role of these cells in bronchial-associated lymphoid tissue formation and B cell homeostasis.

VEGF receptor 2 (KDR) protects airways from mucus metaplasia through a Sox9-dependent pathway.

Mucus-secreting goblet cells are the dominant cell type in pulmonary diseases, e.g., asthma and cystic fibrosis (CF), leading to pathologic mucus metaplasia and airway obstruction. Cytokines including IL-13 are the major players in the transdifferentiation of club cells into goblet cells. Unexpectedly, we have uncovered a previously undescribed pathway promoting mucous metaplasia that involves VEGFa and its receptor KDR. Single-cell RNA sequencing analysis coupled with genetic mouse modeling demonstrates that loss of epithelial VEGFa, KDR, or MEK/ERK kinase promotes excessive club-to-goblet transdifferentiation during development and regeneration. Sox9 is required for goblet cell differentiation following Kdr inhibition in both mouse and human club cells. Significantly, airway mucous metaplasia in asthmatic and CF patients is also associated with reduced KDR signaling and increased SOX9 expression. Together, these findings reveal an unexpected role for VEGFa/KDR signaling in the defense against mucous metaplasia, offering a potential therapeutic target for this common airway pathology.

Airway basal stem cells generate distinct subpopulations of PNECs.

SUMMARYPulmonary neuroendocrine cells (PNECs) have crucial roles in airway physiology and immunity by producing bioactive amines and neuropeptides (NPs). A variety of human diseases exhibit PNEC hyperplasia. Given accumulated evidence that PNECs represent a heterogenous population of cells, we investigate how PNECs differ, whether the heterogeneity is similarly present in mouse and human cells, and whether specific disease involves discrete PNECs. Herein, we identify three distinct types of PNECs in human and mouse airways based on single and double positivity for TUBB3 and the established NP markers. We show that the three PNEC types exhibit significant differences in NP expression, homeostatic turnover, and response to injury and disease. We provide evidence that these differences parallel their distinct cell of origin from basal stem cells (BSCs) or other airway epithelial progenitors.

Cellular and molecular mechanisms of pulmonary malformations

Until now, the cellular and molecular mechanisms of the development of lung defects remain a poorly studied area of pulmonology. In the occurrence of anomalies in the airways of the lungs, a change in the expression of proteins that control early lung morphogenesis in normal conditions (proteins FGF, TGF, SHH, WNT) was established. Along with this, bronchial markers and markers of type 2 alveolocytes play a certain role in the occurrence of lung defects. A number of congenital malformations are caused by improper formation of the airways, which may be associated with the influence of various soluble factors, receptors, transcription factors and microRNAs. The possible role of the pulmonary neuroendocrine system (apudocytes and neuroepithelial bodies) in the pathogenesis and pathobiology of childhood lung diseases, including congenital lung diseases, is discussed.

The Pulmonary NEB ME Is a Complex Intraepithelial Unit.

Among the intrapulmonary myelinated vagal sensory airway receptors, pulmonary neuroepithelial bodies (NEBs) definitely reveal the most complex organisation. This updated review aims at delivering the broad and thorough knowledge of the system that is essential for understanding its physiological relevance.Complexly organised pulmonary NEBs are an integral part of the intrapulmonary airway epithelium of all air-breathing vertebrates. For decades, a quest has been going on to unravel the functional significance of these intriguing structures that appear to be modified in the course of many pulmonary diseases.The pulmonary NEB microenvironment (ME) is composed of organoid clusters of pulmonary neuroendocrine cells (PNECs) that are able to store and release neurotransmitters and are closely contacted by extensive (mainly afferent) nerve terminals, emphasising a potential receptor/effector role and probable signalling to the central nervous system. PNECs are largely shielded from the airway lumen by a special type of Clara cells, the Clara-like cells, with potential stem cell characteristics.Since pulmonary NEBs are widely dispersed in the airway epithelium, and represent less than one percent of the cells in the epithelial lining, investigating the NEB ME largely depends on its unequivocal microscopic identification. Nowadays, multidisciplinary approaches allow to combine functional morphological investigations in cryosections and cleared whole lungs, live cell imaging in lung vibratome slices, and selective gene expression analysis after laser microdissection of genetically tagged NEBs.So far, functional studies of the pulmonary NEB ME revealed that PNECs can be activated by various mechanical and chemical stimuli, resulting in a calciummediated release of neurotransmitters. A number of publications in the past decades have exposed NEBs as potential hypoxia sensors.In the past few years, combination of in vivo experiments and gene expression analysis unveiled the pulmonary NEB ME as a quiescent stem cell niche in healthy postnatal mouse lungs. The stem cell population was shown to be activated by transient mild inflammation, and silencing appears to involve bone morphogenetic protein signalling that may be mediated by vagal afferents.Today, it is clear that only an integrated approach that takes all current information into account will be able to explain the full role of the pulmonary NEB ME in health and disease.

Pulmonary Sensory Receptors.

Among the intrapulmonary myelinated vagal sensory airway receptors, pulmonary neuroepithelial bodies (NEBs) definitely reveal the most complex organisation. This updated review aims at delivering the broad and thorough knowledge of the system that is essential for understanding its physiological relevance.

Correction to: The Pulmonary Neuroepithelial Body Microenvironment.

Correction to: The Pulmonary Neuroepithelial Body Microenvironment.