The rare villin-expressing cell of the murine lower airway is a neuroendocrine cell producing the chemokine C-X-C motif ligand 13 under homeostatic conditions

Abstract

The rare epithelial cell types in the airways are ionocytes, solitary cholinergic chemosensory cells, solitary and clustered neuroendocrine cells. Previous studies reported an enigmatic rare cell in the mouse tracheal epithelium displaying an apical tuft of villin-immunoreactive microvilli. This cell has a morphology similar to cholinergic chemosensory cells. Unlike cholinergic chemosensory cells, the transcription factor Pou2f3 is not required for the development of these unknown cells. Furthermore, some scattered CXCL13 (B-cell attracting chemokine) -immunoreactive cells have been described in the airway epithelium under homeostatic condition, but their identity remained undefined. The present study aimed to clarify the identity of these ill-defined cell types and the role of the transcription factor Pou2f3 in regulation of their cell numbers. To address these aims, appropriate mouse strains, either wild-type or genetically modified, were selected and studied by electron microscopy, immunohistochemistry and RT-PCR. In addition, in silico-analysis was done using publicly available sequencing data sets. The present study identified the enigmatic non-cholinergic chemosensory and villin-expressing cells in the lower airways as a cell population hidden amongst the neuroendocrine cells. In contrast, villin is not expressed in tracheal cholinergic chemosensory cells. The B cell attracting chemokine CXCL13 appeared as a novel mediator with high expression in a subpopulation of neuroendocrine cells, including villin-expressing neuroendocrine cells. CXCL13 is expressed 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- and Vil1-mRNA. Unbiased in silico-analysis of publicly available sequencing data sets did not yield subclusters of neuroendocrine cells. Loss of even just one allele of Pou2f3 results in hyperplasia of neuroendocrine cell phenotypes. The present data demonstrate phenotypic heterogeneity of airway neuroendocrine cells and point towards a putative regulatory role of these cells in B cell homeostasis. The transcription factor Pou2f3 plays a hitherto unrecognized role in their population dynamics.