Hyperplasia and hypertrophy of pulmonary neuroepithelial bodies, presumed airway hypoxia sensors, in hypoxia-inducible factor prolyl hydroxylase-deficient mice

Abstract

Pulmonary neuroepithelial bodies (NEBs), presumed polymodal airway sensors, consist of innervated clusters of amine (serotonin) and peptide-producing cells. While NEB responses to acute hypoxia are mediated by a membrane-bound O2 sensor complex, responses to sustained and/or chronic hypoxia involve a prolyl hydroxylase (PHD)–hypoxia-inducible factor-dependent mechanism. We have previously reported hyperplasia of NEBs in the lungs of Phd1−/− mice associated with enhanced serotonin secretion. Here we use a novel multilabel immunofluorescence method to assess NEB distribution, frequency, and size, together with the number and size of NEB cell nuclei, and to colocalize multiple cytoplasmic and nuclear epitopes in the lungs of Phd1−/−, Phd2+/−, and Phd3−/− mice and compare them with wild-type controls. To define the mechanisms of NEB cell hyperplasia, we used antibodies against Mash1 and Prox1 (neurogenic genes involved in NEB cell differentiation/maturation), hypoxia-inducible factor-1alpha, and the cell proliferation marker Ki67. Morphometric analysis of (% total lung area) immunostaining for synaptophysin (% synaptophysin), a cytoplasmic marker of NEB cells, was significantly increased in Phd1−/− and Phd3−/− mice compared to wild-type mice. In addition, NEB size and the number and size of NEB nuclei were also significantly increased, indicating that deficiency of Phds is associated with striking hyperplasia and hypertrophy of NEBs. In Phd2+/− mice, while mean % synaptophysin was comparable to wild-type controls, the NEB size was moderately increased, suggesting an effect even in heterozygotes. NEBs in all Phd-deficient mice showed increased expression of Mash1, Prox1, Ki67, and hypoxia-inducible factor-1alpha, in keeping with enhanced differentiation from precursor cells and a minor component of cell proliferation. Since the loss of PHD activity mimics chronic hypoxia, our data provide critical information on the potential role of PHDs in the pathobiology and mechanisms of NEB cell hyperplasia that is relevant to a number of pediatric lung disorders.

Pulmonary neuroendocrine cells: physiology, tissue homeostasis and disease

“…In addition, PNECs express prolyl hydroxylase domain enzymes (PHDs), which respond to hypoxia by catalyzing the hydroxylation of hypoxia-inducible factor 1 (HIF-1; Box 1 ), thus stabilizing it ( Semenza, 2009 ). Importantly, loss of PHDs provokes a PNEC hyperplasia phenotype ( Pan et al, 2012 , 2016 ). The role of HIF-1 in PNEC differentiation could be another interesting topic for future studies.…”

HIF Oxygen Sensing Pathways in Lung Biology

“…Cell proliferative responses to hypoxia are not restricted to club cells in the bronchial epithelium; they have also been found in the pulmonary neuroepithelial bodies (NEBs). These structures are presumed airway sensors, made-up of innervated clusters of amine (serotonin) and peptide-producing cells that reside in the bronchial epithelium [ 56 , 57 ]. In humans and in animal models, hypoxia leads to hyperplasia of NEBs, and PHD1 and PHD3 have been implicated in this response [ 56 , 58 , 59 ].…”

“…These structures are presumed airway sensors, made-up of innervated clusters of amine (serotonin) and peptide-producing cells that reside in the bronchial epithelium [ 56 , 57 ]. In humans and in animal models, hypoxia leads to hyperplasia of NEBs, and PHD1 and PHD3 have been implicated in this response [ 56 , 58 , 59 ]. Indeed, Phd1 - and Phd3 -deficient mice show marked hypertrophia and NEB proliferation, in parallel to the induction of HIF1α in some of synaptophysin (SYP) positive cells typically found in NEBs [ 56 , 59 ].…”

“…In humans and in animal models, hypoxia leads to hyperplasia of NEBs, and PHD1 and PHD3 have been implicated in this response [ 56 , 58 , 59 ]. Indeed, Phd1 - and Phd3 -deficient mice show marked hypertrophia and NEB proliferation, in parallel to the induction of HIF1α in some of synaptophysin (SYP) positive cells typically found in NEBs [ 56 , 59 ]. However, it is unclear whether or not HIF1 contributes to NEB proliferation in response to hypoxia and whether NEB hyperproliferation is driven by HIF2, as in club cells.…”

Mapping the Central and Peripheral Projections of Lung Innervating Sensory Neurons

“…Despite their rarity, PNECs are frequent targets of vagal sensory nerves. They are airway sensors that are activated following allergen challenge 52,53 , mechanical stretch 54,55 and hypoxia 56,57 . Our labeling with Trpv1-cre confirmed previous finding in the rat lung that these nerves contact the basal surface of NEBs 58,59 .…”