Pulmonary neuroendocrine cells function as airway sensors to control lung immune response

Abstract

The lung is constantly exposed to environmental atmospheric cues. How it senses and responds to these cues is poorly defined. Here, we show that Roundabout receptor (Robo) genes are expressed in pulmonary neuroendocrine cells (PNECs), a rare, innervated epithelial population. Robo inactivation in mouse lung results in an inability of PNECs to cluster into sensory organoids and triggers increased neuropeptide production upon exposure to air. Excess neuropeptides lead to an increase in immune infiltrates, which in turn remodel the matrix and irreversibly simplify the alveoli. We demonstrate in vivo that PNECs act as precise airway sensors that elicit immune responses via neuropeptides. These findings suggest that the PNEC and neuropeptide abnormalities documented in a wide array of pulmonary diseases may profoundly affect symptoms and progression.

Fig. 1. Robo mutants exhibit alveolar simplification and heightened immune response

(A and B) Hematoxylin and eosin (H&E) staining of alveolar region at P22. (C) qRT-PCR of P7 lungs. (D and E) IsoB4 labeling of macrophages at P22. (F and G) X-Gal–stained heterozygous Robo1^lacZ/+;Robo2^lacZ/+ lungs. (F) P0, arrows indicate expression in PNECs. (G) E15.5, with anti-CGRP immunostaining (red). *P < 0.05; **P < 0.001.

Fig. 2. Robo1;2 are required for PNEC clustering

Immunostained vibratome lung slices. (A and B) ASCL1 immunostaining labels nascent PNECs. (C to F) Synaptophysin immunostaining labels differentiated PNECs and their associated nerves. Arrowheads indicate solitary PNECs, arrows indicate clustered PNECs in NEBs, and asterisks indicate airway lumen. Scale bars, 30 µm.

Fig. 3. Increase in neuropeptide level contributes to increased macrophage infiltration

(A) qRT-PCR of PNEC peptide transcript levels in P5 lungs. *P < 0.05, **P < 0.001. (B to E) IsoB4 labeling of macrophages at P22. Scale bars, 40 µm. (F) Quantification presented as the relative percentage of macrophage to total cell ratio normalized to Shhcre; Robo^+/−;Cgrp^+/+. **P < 0.01; ***P < 0.0001; n.s., not significantly different (P ≥ 0.05).

Fig. 4. Macrophage reduction by clodronate treatment attenuates alveolar simplification

(A to D) IsoB4 labeling of macrophages at P22. Scale bar, 50 µm. (E) Macrophage quantification as the relative percentage of macrophage to total cell ratio normalized to control mice with liposome control treatment. (F to I) H&E staining of alveolar region at P22. Scale bar, 100 µm. (J) Quantification of mean linear intercept (MLI). ***P < 0.0001; n.s., not significantly different (P ≥ 0.05).