Abstract
Background
The microenvironment (ME) of neuroepithelial bodies (NEBs) harbors densely innervated groups of pulmonary neuroendocrine cells that are covered by Clara-like cells (CLCs) and is believed to be important during development and for adult airway epithelial repair after severe injury. Yet, little is known about its potential stem cell characteristics in healthy postnatal lungs.
Methods
Transient mild lung inflammation was induced in mice via a single low-dose intratracheal instillation of lipopolysaccharide (LPS). Bronchoalveolar lavage fluid (BALF), collected 16 h after LPS instillation, was used to challenge the NEB ME in ex vivo lung slices of control mice. Proliferating cells in the NEB ME were identified and quantified following simultaneous LPS instillation and BrdU injection.
Results
The applied LPS protocol induced very mild and transient lung injury. Challenge of lung slices with BALF of LPS-treated mice resulted in selective Ca2+-mediated activation of CLCs in the NEB ME of control mice. Forty-eight hours after LPS challenge, a remarkably selective and significant increase in the number of divided (BrdU-labeled) cells surrounding NEBs was observed in lung sections of LPS-challenged mice. Proliferating cells were identified as CLCs.
Conclusions
A highly reproducible and minimally invasive lung inflammation model was validated for inducing selective activation of a quiescent stem cell population in the NEB ME. The model creates new opportunities for unraveling the cellular mechanisms/pathways regulating silencing, activation, proliferation and differentiation of this unique postnatal airway epithelial stem cell population.
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Background
The postnatal lung is a conditionally renewing organ with a very low airway epithelial cell turnover in the absence of injury, with less than 1 % of cells dividing at any time point in several species [1, 2]. However, the lungs and airways are capable of rapidly increasing regeneration rate to replace damaged tissue, with local stem and progenitor cells re-entering the cell cycle (for reviews see [3, 4]). Adult stem cells are defined as rare cells present in different niches, with a high proliferative potential and a lifelong ability to self-renew, maintain a variety of cell populations in the steady state and/or replace damaged cells following injury [3, 5, 6].
Neuroepithelial bodies (NEBs) occur in the airway epithelium as densely innervated clusters of pulmonary neuroendocrine cells (PNECs; for review see [7]). In many species (including humans) PNECs are covered by Clara-like cells (CLCs), leaving only thin apical processes of PNECs in contact with the airway lumen. CLCs, PNECs and their extensive innervation together constitute the so-called ‘NEB microenvironment (NEB ME)’ [8,9,10,11]. CLCs have also been referred to as variant Clara cell secretory protein (CCSP)-expressing cells (vCE cells) [12].
The clusters of PNECs release bioactive substances upon stimulation [13,14,15,16,17,18] and are selectively contacted by mainly vagal afferent nerve terminals [9, 19]. Pulmonary NEBs should therefore be regarded as complex intraepithelial sensory airway receptors, capable of sensing and transducing hypoxic, mechanical, chemical and likely also other stimuli [14, 15, 20](for reviews see [9, 21,22,23]).
Apart from being airway sensors, NEBs may fulfill some other proposed physiological roles in the airways during fetal and perinatal life [21, 22, 24,25,26]. The relatively large number of NEBs encountered in the prenatal lung has been explained by their potential role in the regulation of bronchogenesis, as PNECs represent the first cell type that differentiates during embryonic lung development [27]. The possible paracrine regulation of embryonic airway epithelial cell growth by NEBs has been proposed more than 25 years ago based on cell proliferation studies, illustrating that the number of labeled divided cells progressively decreases with increasing distance from NEBs [28].
Throughout the past decade, the NEB ME has been put forward as one of the potential stem cell sources/niches that are dispersed along the epithelial lining of the mammalian respiratory tract [29,30,31,32].
The suggested stem cell capacities of the NEB ME in healthy postnatal mouse lungs were recently confirmed using an optimized laser microdissection (LMD) protocol that allows for the selective collection of high quality mRNA samples of the NEB ME [33]. Expression analysis of an extensive panel of genes, selected for their involvement in cell development, proliferation and stem cell signaling, enabled to define a stem cell ‘signature’ for the NEB ME, an indication that the NEB ME may indeed represent a functional stem cell niche in healthy postnatal mouse airways [33].
Both cell types in the NEB ME, i.e., PNECs and CLCs, have been proposed as potential airway epithelial progenitor cells [12, 34,35,36,37]. The observation that NEBs, or at least epithelial cell groups with similar characteristics, show hyperplasia in many airway diseases/disorders [38,39,40], and seem to play a role as precursors for small cell lung carcinoma (SCLC) [6, 34, 41], evidently suggests a role for PNECs as airway epithelial progenitors. However, the ‘stemness’ of PNECs is currently questioned since PNECs on their own were not able to restore the airway epithelium after ablation of both Clara cells (CCs) and CLCs [12]. On the other hand, self-renewing and stem cell characteristics have been assigned to CLCs/vCE cells based on lineage-tracing analysis in murine models [12, 42]. During embryonic development, cells surrounding PNECs, i.e., presumptive CLCs, remain undifferentiated [30]. CLCs/vCE cells appear to be resistant to naphthalene ablation because, unlike CCs, they do not express the cytochrome P450 2F2 isozyme [12, 43, 44]. It has been reported that CLCs in postnatal lungs show the capacity to regenerate both CCs and ciliated cells [30, 64,65,66,67]. Lineage-tracing models suggest that CLCs have the capacity to self-renew [12, 42]. Clara cell-like precursors appear to generate both Clara and ciliated cells during development and repair, driven by Notch signaling [77], is challenged by the notion that repair of severe airway injury is associated with hyperplasia of PNECs [35]. Chemically or genetically induced full depletion of CCs revealed a typical proliferation of PNECs [36, 70]. Although at least subpopulations of PNEC-like progenitors are believed to give rise to CCs and even to alveolar epithelial cells during early development [30, 78, 79], most studies suggest that PNECs are not able to restore adult airway epithelium after ablation of all types of CCs [12]. Elimination of PNECs prior to CC depletion seems to have no apparent consequence for CC regeneration [34], but the same study reports that to some extent PNECs may contribute to CCs and ciliated cells following severe lung injury.
The presented data (LPS treatment; 48 h experimental window) show a very low number of divided PNECs in the NEB ME, which is not significantly different between LPS-challenged, sham and untreated control mice. In contrast to the well-illustrated proliferation of endocrine cells (PNECs) in addition to CLCs/vCE cells in several studies that are based on a full depletion of CCs [12, 35, 70], our LPS-based transient mild injury model for the selective proliferation of CLCs offers the important advantage that PNECs in the NEB ME are not affected by the procedure. The latter is in agreement with our observation that soluble mediators in BALF of LPS-challenged mice result in a calcium-mediated activation of CLCs but not of PNECs in the NEB ME in lung slices of control mice.
Certainly, PNECs can secrete regulatory factors –e.g. gastrin-releasing peptide (bombesin) and CGRP, potential epithelial cell mitogens [80]– that may support and regulate airway epithelial cell renewal/proliferation and differentiation. PNECs, however, are not only able to produce, store and secrete a variety of bioactive substances –some of which may directly influence CLCs [13]– but also to monitor calcium-mediated events in surrounding CLCs [14], and may therefore be involved in creating a niche to maintain the stem cell characteristics of CLCs.
Conclusion
Based on a single low-dose intratracheal LPS instillation, a highly reproducible and minimally invasive lung inflammation model was generated and validated for inducing selective activation of a quiescent airway stem cell population –the so-called CLCs/vCE cells– in the NEB ME.
Important advantages compared to earlier models, which were mainly based on full ablation of CCs, are the absence of both severe epithelial injury and additional proliferation of endocrine cells (PNECs).
The fact that CLCs in the NEB ME can be activated from a silent to a dividing stem cell population in the absence of severe airway epithelial damage creates new opportunities for unraveling the cellular mechanisms/pathways regulating silencing, activation, proliferation and differentiation of this unique postnatal airway epithelial stem cell population.
The presented data are supportive of potentially important selective roles of the postnatal airway stem cell niche of the NEB ME, and enable the identification of pathways that should allow uncoupling of essential repair mechanisms from severe lung injury and inflammation.
Abbreviations
- [Ca2+]i :
-
Intracellular calcium concentration
- [K+]o :
-
Extracellular potassium concentration
- 4-Di-2-ASP:
-
4-(4-diethylaminostyryl)-N-methylpyridinium iodide
- BALF:
-
Bronchoalveolar lavage fluid
- BrdU:
-
5-bromo-2′-deoxyuridine
- BSA:
-
Bovine serum albumin
- BW:
-
Bodyweight
- CAE:
-
Control airway epithelium
- CC:
-
Clara cell
- CCSP:
-
Clara cell secretory protein
- CGRP:
-
Calcitonin gene-related peptide
- CLC:
-
Clara-like cell
- DMEM-F-12:
-
Dulbecco’s modified Eagle’s medium/F-12
- EIP:
-
End-inspiratory pause
- GAD67:
-
Glutamic acid decarboxylase 67
- H&E:
-
Hematoxylin and eosin
- i.p.:
-
Intraperitoneal
- LCI:
-
Live cell imaging
- LMD:
-
Laser microdissection
- LPS:
-
Lipopolysaccharide
- Mc:
-
Monoclonal
- ME:
-
Microenvironment
- NEB ME:
-
Neuroepithelial body microenvironment
- NEB:
-
Neuroepithelial body
- PBS:
-
Phosphate-buffered saline
- Pc:
-
Polyclonal
- PD:
-
Postnatal day
- PF:
-
Paraformaldehyde
- PNEC:
-
Pulmonary neuroendocrine cell
- ROI:
-
Region of interest
- RT:
-
Relaxation time
- SCLC:
-
Small cell lung carcinoma
- SEM:
-
Standard error of means
- Te:
-
Expiratory time
- TV:
-
Tidal volume
- UP1:
-
Urine protein 1
- vCE:
-
Variant CCSP-expressing
- WT-Bl6:
-
Wild type C57BL/6 J
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Acknowledgements
The authors wish to thank Dominique De Rijck, Robrecht Lembrechts, Carmen Rottiers, Francis Terloo, Elien Theuns, Sofie Thys and Danny Vindevogel for their assistance.
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This study was financially supported by a GOA BOF 2015 grant (No. 30729) of the University of Antwerp.
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LV developed and carried out the experiments and prepared the manuscript. DA and IB designed the experiments, supervised the analysis and edited the manuscript. All authors regularly discussed the experiments and data, commented on the text, and read and approved the submitted manuscript.
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National and international principles of laboratory animal care were followed, and experiments were approved by the local animal ethics committee of the University of Antwerp (ECD 2014–66 and 2017–49).
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Verckist, L., Pintelon, I., Timmermans, JP. et al. Selective activation and proliferation of a quiescent stem cell population in the neuroepithelial body microenvironment. Respir Res 19, 207 (2018). https://doi.org/10.1186/s12931-018-0915-8
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DOI: https://doi.org/10.1186/s12931-018-0915-8