Cytokines reprogram airway sensory neurons in asthma

Nociceptor neurons play a crucial role in maintaining the body’s homeostasis by detecting and responding to potential dangers in the environment. However, this function can be detrimental during allergic reactions, since vagal nociceptors can contribute to immune cell infiltration, bronchial hypersensitivity, and mucus imbalance, in addition to causing pain and coughing. Despite this, the specific mechanisms by which nociceptors acquire pro-inflammatory characteristics during allergic reactions are not yet fully understood. In this study, we aimed to investigate the molecular profile of airway nociceptor neurons during allergic airway inflammation and identify the signals driving such reprogramming. Using retrograde tracing and lineage reporting, we identified a unique class of inflammatory vagal nociceptor neurons that exclusively innervate the airways. In the ovalbumin mouse model of airway inflammation, these neurons undergo significant reprogramming characterized by the upregulation of the NPY receptor Npy1r. A screening of cytokines and neurotrophins revealed that IL-1β, IL-13 and BDNF drive part of this reprogramming. IL-13 triggered Npy1r overexpression in nociceptors via the JAK/STAT6 pathway. In parallel, sympathetic neurons and macrophages release NPY in the bronchoalveolar fluid of asthmatic mice, which limits the excitability of nociceptor neurons. Single-cell RNA sequencing of lung immune cells has revealed that a cell-specific knockout of Npy1r in nociceptor neurons in asthmatic mice leads to an increase in airway inflammation mediated by T cells. Opposite findings were observed in asthmatic mice in which nociceptor neurons were chemically ablated. In summary, allergic airway inflammation reprograms airway nociceptor neurons to acquire a pro-inflammatory phenotype, while a compensatory mechanism involving NPY1R limits nociceptor neurons’ activity. Mice in which NPY1R was floxed in nociceptor neurons (sample 1;3;5; n=3) and littermate controls (samples 2;4;6; n=3), as well as C57BL6 mice depleted of peptidergic nociceptors with RTX (samples 14;16; n=2) or not (samples 13;15; n=2) underwent the classic ovalbumin asthma protocol (samples 1-6 and 13-16). C57BL6 mice treated with PBS were used for naive controls (sample 9; n=1). Mice lungs were harvested (day 18), minced with a razor blade, digested in Collagenase IV (1.6mg/ml) + DNAse I (10ug/ml) in DMEM buffer supplemented with Hepes (10mM) for 30 min. Lungs were triturated with a 1mL pipette and cell suspensions were filtered (40um). 2 different mice were pooled for each sample. RBC were lysed before staining with viability dye and anti-CD45. 500000 live immune cells were sorted on a FACS ARIA III (BD) and harvested in collection buffer (PBS+ 0.04% BSA + 50% FBS). Cells were then fixed, before single cell library preparation using Chromium 10x fixed RNA kits and instructions (#PN-1000414 and #PN-1000497). Libraries were subsequently sequenced on Illumina Novaseq X.