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.

NEUROEPITHELIAL BODIES OF THE RAT’S LUNGS IN EXPERIMENTAL ASTHMA

The epithelium of the respiratory tract of mammalian lungs contains pulmonary neuroendocrine cells, represented by both single cells and innervated clusters forming neuroepithelial bodies (NEB). Since NEB are intensively innervated and produce highly specific biologically active substances, such as the bronchoconstrictor serotonin, the level of which increases during hypoxia, it is assumed that these structures can play a key role in the pathogenesis of bronchial asthma (BA).The purpose of this study was to the detection and analysis of NEB in the lungs with experimental BA.For the study, we used the lungs of sexually mature Wistar rats (n = 5). NEB was detected by monoclonal antibodies to synaptophysine.It has been found but that in the context of experimental asthma 76.6% NEB were located as part of a simple cuboidal epithelium of small bronchi and respiratory bronchioles. 17.6% of NEB were found in the composition of the epithelial layer and only 5.8% are single NEB in the composition of the epithelium of the small bronchi. At the same time, a greater number of NEB localized in the bronchi were composed of 6 cells (46.2%), 38.5% of 4 cells, and 15.3% of more than 10 cells in one cluster. As in our previous studies, most of the NEB were located in the vicinity of the synaptophysi-immunopositive terminals. Against the background of asthma occurred reduction in the number of large clusters NEB and increased concentrations of medium size of NEB. The results obtained indicate the effect of inflammation on the functional features of the neuroendocrine system of the lungs and the possible contribution of NEB to the inflammatory cascade in BA.

Pulmonary neuroepithelial bodies are polymodal airway sensors: Evidence for CO2/H+ sensing

Pulmonary neuroepithelial bodies (NEB) in mammalian lungs are thought to function as airway O2 sensors that release serotonin (5-HT) in response to hypoxia. Direct evidence that NEB cells also respond to airway hypercapnia/acidosis (CO2/H+) is presently lacking. We tested the effects of CO2/H+ alone or in combination with hypoxia on 5-HT release from intact NEB cells in a neonatal hamster lung slice model. For the detection of 5-HT release we used carbon fiber amperometry. Fluorescence Ca2+ imaging method was used to assess CO2/H+-evoked changes in intracellular Ca2+. Exposure to 10 and 20% CO2 or pH 6.8–7.2 evoked significant release of 5-HT with a distinct rise in intracellular Ca2+ in hamster NEBs. This secretory response was dependent on the voltage-gated entry of extracellular Ca2+. Moreover, the combined effects of hypercapnia and hypoxia were additive. Critically, an inhibitor of carbonic anhydrase (CA), acetazolamide, suppressed CO2/H+-mediated 5-HT release. The expression of mRNAs for various CA isotypes, including CAII, was identified in NEB cells from human lung, and protein expression was confirmed by immunohistochemistry using a specific anti-CAII antibody on sections of human and hamster lung. Taken together our findings provide strong evidence for CO2/H+ sensing by NEB cells and support their role as polymodal airway sensors with as yet to be defined functions under normal and disease conditions.