Airway basal stem cells generate distinct subpopulations of PNECs

Abstract

Pulmonary neuroendocrine cells (PNECs) have crucial roles in airway physiology and immunity by producing bioactive amines and neuropeptides (NPs). A variety of human diseases exhibit PNEC hyperplasia. Given accumulated evidence that PNECs represent a heterogenous population of cells, we investigate how PNECs differ, whether the heterogeneity is similarly present in mouse and human cells, and whether specific disease involves discrete PNECs. Herein, we identify three distinct types of PNECs in human and mouse airways based on single and double positivity for TUBB3 and the established NP markers. We show that the three PNEC types exhibit significant differences in NP expression, homeostatic turnover, and response to injury and disease. We provide evidence that these differences parallel their distinct cell of origin from basal stem cells (BSCs) or other airway epithelial progenitors.

Keywords: 5-HT; BSC; NEB; NEHI; NP; PNEC; SIDS; TUBB3; cellular protrusion.

Figure 1.. Identification of TUBB3⁺ PNECs in the human lung

(A–E) Human airways from healthy donors, sized from 2–3 mm in diameter to bronchioles, were double stained for TUBB3 and specified markers of airway epithelial cells and neurons (NeuNs) in (A), GRP in (B) and (D), and ASCL1 in (C). Arrowhead marks single GRP⁺ PNECs. Arrows mark GRP⁺TUBB3⁺ PNECs. Asterisks mark single TUBB3⁺ PNECs. Images in (C) are from two adjacent sections. The dotted lines in (B)–(D) mark basement membrane beneath the airway epithelium. (E) Quantification of the three types of PNECs in healthy donor lungs. (F) Representative images of double staining for GRP and TUBB3 in a patient with NEHI. Arrows mark GRP⁺TUBB3⁺ PNECs. Background green fluorescence was caused by red blood cells. (G–J) Representative images of CHGA and TUBB3 staining of adjacent sections from SIDS cases (G). Arrows mark CHGA⁺TUBB3⁺ PNECs. Data were quantified as the percentage of PNECs that were NP⁺TUBB3⁺ in (H), the number of single NP⁺ NEBs in (I), and NP⁺TUBB3⁺ NEBs in (J) per millimeter of airway epithelium in non-SIDS controls (n = 4), high 5-HT SIDS cases (n = 4), and normal 5-HT SIDS cases (n = 5). NEBs with more than three PNECs were counted. (K) Representative images of staining for TUBB3 (arrow) and Ki67 (arrowhead) on adjacent sections from SIDS cases. Bar graphs represent means ± SEM. *p < 0.05, **p < 0.01 by Student’s t test. See also Figure S1 and Table S1.

Figure 2.. Characterization of the three PNEC types in the trachea and the intrapulmonary airway in mice

(A) Representative images of CGRP and TUBB3 staining in the intrapulmonary airway. The outlined area is shown in the bottom panel. n = 6. (B) Relative abundance of the three PNEC types in the intrapulmonary (intra) airway and the trachea. n = 3 mice; five sections from each mouse. (C) Representative images of double staining of mouse tracheal sections for TUBB3 and markers of epithelial cells. Arrows mark CGRP⁺TUBB3⁺ PNECs. Asterisk marks a single TUBB3⁺ PNEC. n = 6. (D) Scheme of neuroendocrine cell lineage tracing using Ascl1-CreERT2;Rosa(tmRed) mice. (E–H) Representative images of CGRP staining of the intrapulmonary airway (E) and the trachea (G) in TAM-treated, Ascl1-CreERT2;Rosa(tmRed) mice on D7 and D97. Arrow marks tmRed⁺ PNECs. Data were quantified in (F) and (H). n = 3 mice, 3–5 sections from each mouse. See also Figure S2.

Figure 3.. BSCs generate TUBB3⁺ PNECs in culture

(A) Scheme of BSC lineage tracing in TAM-inducible, p63-CreERT2;Rosa(tmRed) mice. (B and C) The trachea (B) and the intrapulmonary airway (C) were analyzed using 6–12 fields (20×) per sample and a total of three mice. See also Figures S3 and S4. (D) Scheme of PNEC lineage labeling of the Ascl1-CreERT2;Rosa(tmRed) BSC culture. The culture was treated with 4-OH-TAM on day 15 ALI and analyzed on days 17–19 of ALI. (E) Relative abundance of the three PNEC types in ALI culture. (F) Scheme of human BSC culture in ALI. (G and H) Day 14 ALI culture was analyzed by staining for TUBB3 and the specified markers. In (B) and (G), arrows show GRP⁺TUBB3⁺ PNECs, and asterisks mark single TUBB3⁺ cells. Data are representative results using BSCs from three donors.

Figure 4.. TUBB3 is required for PNECs to form cellular protrusions

(A and C) Representative images of double staining for CHGA and TUBB3 in ALI culture of wild-type and Tubb3^−/− BSCs. Asterisks mark “snail” PNECs with very short protrusions. (B and D) Relative abundance of CHGA⁺ PNECs with protrusions of different lengths (B) and patterns (D) in wild-type and Tubb3^−/− cultures. More than 500 PNECs were counted in each experiment, with a total of two independent experiments. (E) 5-HT secretion in ALI culture of wild-type and Tubb3^−/− BSCs by ELISA. Cultures in triplicates and from two independent experiments were assayed. (F) Representative images of PNECs in the trachea of wild-type and Tubb3^−/− mice. An asterisk marks a snail PNEC. (G–I) Quantification of PGP9.5⁺ PNECs in (G), snail PNECs in (H), and p63⁺ BSCs in (I) per millimeter along the tracheal epithelium in wild-type and Tubb3^−/− mice. (J) Representative images of CGRP⁺ NEBs in the intrapulmonary airway of wild-type and Tubb3^−/− mice. n = 15 sections from three mice of each genotype. Bar graphs represent means ± SEM. *p < 0.05 by Student’s t test. Scale bars, 50 μm.