The lung, with its vast surface area, senses and responds to signals in inhaled air. Aberrant interactions between the lung and the environment underlie many diseases, including asthma. In vitro data show that pulmonary neuroendocrine cells (PNECs), a rare airway epithelial cell population, can act as chemosensors. Once stimulated in culture, they release dense core vesicles rich in neuropeptides, amines, and neurotransmitters. These bioactive molecules are capable of eliciting immune and physiological responses. A recent in vivo study by our group revealed that the proper development of PNECs into self-clustering units called neuroepithelial bodies is essential for restricting the number of immune cells in the naïve lung. However, whether PNECs can function in vivo to translate exogenous airway signals such as allergens into the cascade of downstream responses is unknown.

To test the hypothesis that PNECs act as sensors in the lung, we generated mouse mutants that lack PNECs by inactivating Ascl1 in the airway epithelium—i.e., mutants that were depleted of PNECs starting at development. We exposed these mutants to either ovalbumin or house dust mites, following regimes of existing asthma models. We determined whether the mutants showed different asthmatic responses than controls. We elucidated the underlying mechanisms by identifying molecular effectors and cellular targets of PNECs. To complement the functional tests in mice, we investigated whether human asthma patients showed pathological changes in their PNECs.

Although normal at baseline, Ascl1-mutant mice exhibited severely reduced goblet cell hyperplasia and immune cell numbers compared with controls after allergen challenge. In investigating possible molecular effectors, we found that several PNEC products were decreased in mutants relative to controls after allergen challenge, including calcitonin gene-related peptide (CGRP) and γ-aminobutyric acid (GABA). In exploring possible cellular targets, we found that innate lymphoid group 2 cells (ILC2s) were enriched at airway branch points, similar to PNECs. The PNEC product CGRP stimulated ILC2 production of interleukin-5 in culture. Conversely, inactivation of the CGRP receptor gene Calcrl in ILC2s led to dampened immune responses to allergens. In contrast to CGRP, GABA did not increase ILC2 cytokine secretion. Rather, inactivation of GABA biogenesis led to defective goblet cell hyperplasia after allergen challenge, suggesting that GABA is required for this response in the airway epithelium. The instillation of a mixture of CGRP and GABA in Ascl1 mutants restored both immune cell increases and goblet cell hyperplasia after allergen challenge, indicating that these products are the primary molecular effectors of PNECs in vivo. Consistent with these results from mice, we found increased PNEC numbers and cluster sizes in human asthma patients, which may underlie the heightened response to allergens in these individuals.

Our results demonstrate that PNECs, despite being a rare population of cells in the airway, are critical for amplifying the airway allergen signal into mucosal type 2 responses. Specifically, PNECs act through their product GABA to stimulate airway epithelial mucus production. In parallel, PNECs act through another product, CGRP, to stimulate ILC2 production of cytokines, which in turn recruit downstream immune cells. PNECs and ILC2s form neuroimmunological modules at the airway branch points, which are also the sites where airway particles are enriched. Our findings indicate that the PNEC-ILC2 axis functions to …