Selective Activation and Proliferation of a Unique Stem Cell Population in the Neuroepithelial Body Microenvironment by Transient Acute Lung Injury
Authors: Verckist Line , Rottiers Carmen , Lembrechts Robrecht , Pintelon Isabel , Timmermans Jean‐Pierre , Brouns Inge , Adriaensen Dirk 
Published: November 2023

The microenvironment (ME) of pulmonary neuroepithelial bodies (NEBs) has been suggested to be implicated in airway development and adult airway epithelial repair after severe injury. Regardless the increasing evidence that NEBs act as a stem cell niche under these circumstances, little is known about the potential stem cell characteristics of the NEB ME in healthy postnatal lungs. The NEB ME consists of highly innervated groups of pulmonary neuroepithelial endocrine cells that are specifically covered by a unique population of Clara‐like cells (CLCs) with supposed stem cell characteristics. The relatively low number and widespread distribution of NEBs in the epithelium of intrapulmonary airways of postnatal animals has so far hampered the direct identification of mechanisms and signaling processes to support their characterization as a stem cell niche.

A GAD67‐GFP mouse model, in which NEB cells selectively express GFP, permits their straightforward identification in cryostat sections of postnatal mouse lungs. Transient acute lung injury (ALI) was induced via intratracheal instillation of lipopolysaccharide (LPS; 1mg/kg bw). Lung function was monitored using a noninvasive unrestrained whole body plethysmograph. In one experiment, bronchoalveolar lavage fluid (BALF) was collected 24h after LPS instillation, and used to challenge the NEB ME in ex vivo lung slices of control mice. In another experiment, BrdU (10 mg/kg bw), as a marker for cells that divide during the observation window, was injected intraperitoneally at the time of LPS instillation and after 24h, the animals were euthanized after 48h, and the lungs were processed for freeze sectioning and BrdU immunostaining.

Challenge of lung slices with BALF of ALI mice was shown to induce a reversible and reproducible selective Ca2+‐mediated activation of CLCs in the NEB ME of control mice. 48h after LPS challenge and BrdU injection, lung sections of ALI mice revealed a remarkable and highly significant increase in the number of BrdU‐labeled cells that surround NEBs, as compared to control lungs. As judged from sham‐treated mice, the instillation procedure itself also resulted in a modest increase in NEB‐associated BrdU‐labeled cells. Preliminary plethysmography data revealed that a single intratracheal LPS instillation results in short and transient ALI. Lung function parameters show the largest changes around six hours after treatment, and have restored to values that resemble those of the control group after 24h.

In conclusion, mild transient ALI appears to result in the production of soluble mediators in the airways that selectively activate, and induce proliferation of, a stem cell population in the NEB ME. The changes that occur in the dividing cells will be further explored using gene expression profiling. The described workflow, together with our previously optimized LMD protocol, provides a solid base for unraveling molecular characteristics of the NEB ME.

Support or Funding Information

Hercules Foundation grant AUAH‐09‐001 (DA); UA grant GOA BOF 2015 (DA).