Pulmonary neuroendocrine cell (PNEC) hyperplasia often occurs in lung diseases, including allergic asthma. We previously reported that PNEC-derived calcitonin gene-related peptide (CGRP) likely stimulates group 2 innate lymphoid cells (ILC2), exacerbating asthma phenotypes in a mouse model. Here, we investigate the role of glial cell-line-derived neurotrophic factor (GDNF) and rearranged during transfection (RET) signaling in PNEC hyperplasia and its therapeutic potential in asthma. PNECs expressed GDNF receptors, which were activated primarily by infiltrating inflammatory cells. Application of a RET-specific inhibitor suppressed ILC2 levels, PNEC hyperplasia, and airway allergic responses. We suggest that GDNF-RET signaling promotes PNEC hyperplasia and that the PNEC-CGRP-ILC2 axis is closely associated with the development of allergic asthma, presenting a possible new treatment strategy.

Mice

Specific pathogen-free male C57BL/6N mice were purchased from Japan SLC Inc. and CLEA, Japan. GAD67-green fluorescent protein (GFP) knock-in mice (ICR.Cg-Gad1) were provided by RIKEN BRC and were backcrossed for at least eight generations with C57BL/6 strains. All animal experiments in this manuscript were carried out in accordance with both the ARRIVE guideline for reporting in vivo experiments and the guidelines approved by the Committee of Animal Experiments at the Tohoku Medical and Pharmaceutical University (Approval Numbers: A23015, A24013, and A25031). All experimental animals were bred in a 12-h light-dark cycle environment with free access to food and water except during the experimental period. To minimise pain and suffering, all procedures were performed under appropriate anaesthesia and analgesia. Animals were euthanized when necessary by CO₂ inhalation followed by an overdose of anaesthetic.

Asthma mouse model

An asthma mouse model was established as previously described. Briefly, six- to seven-week-old mice were sensitized by intraperitoneal injection of chicken ovalbumin (OVA; Grade V; Sigma-Aldrich, St Louis, MO, USA; 8 μg per mouse) adsorbed onto aluminum hydroxide (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan; 4 mg per mouse) on days 0 and 5. On days 17 and 24, all mice were challenged with aerosolized OVA (5 mg/mL) to induce an asthma-like phenotype. Pralsetinib (Selleck Bio., Houston, TX, USA) was dissolved in DMSO (Nacalai Tesque, Kyoto, Japan) and diluted in sterile corn oil (Nacalai Tesque). The resulting solution was administered orally by gavage at 200 μL per mouse per dose (10 mg/kg; 5% DMSO and 95% corn oil). Pralsetinib was administered once daily from day 18 for nine consecutive days. A vehicle control group received the corresponding solvent. Bronchoalveolar lavage (BAL) was performed. Briefly, the lungs were lavaged twice by injecting 0.25 mL of cold phosphate-buffered saline (PBS), and the recovered PBS was pooled. BALF samples were processed to count the total cell counts and cell differentials. In some experiments, lungs were harvested from a subset of mice, and homogenates were prepared for RNA and protein extraction. RNA and proteins were prepared for RT–PCR and ELISA analyses, respectively. Schematics of the experimental protocol are shown in Figures 2A and 4A.

Preparation of lung cell suspensions

Lungs from C57BL/6N wild-type and/or GAD67-GFP knock-in mice were excised, minced, and enzymatically digested using the enzyme mixture supplied in the Multi Tissue Dissociation Kit 1 (Miltenyi Biotec, Bergisch Gladbach, Germany). Dissociation was performed using a gentleMACS Dissociator (Miltenyi Biotec) with the 37C_n_LUNG program. The digested material was filtered through 70-µm cell strainers, washed with phosphate-buffered saline (PBS), and resuspended in PBS supplemented with 5 mM EDTA and 1% bovine serum albumin (PEB) buffer. The resulting lung cell suspensions were used to isolate the indicated cell populations by magnetic separation and flow cytometric sorting.

Isolation of CD45⁺, CD146⁺, and CD31⁺ cells

CD45⁺ hematopoietic cells were first isolated from lung cell suspensions using CD45 MicroBeads (Miltenyi Biotec) and an autoMACS Pro Separator (Miltenyi Biotec) with the Possel program. The CD45-depleted fraction was subsequently incubated with biotin-conjugated anti-CD146 antibody (BioLegend, San Diego, CA, USA), followed by Streptavidin MicroBeads (Miltenyi Biotec). CD146⁺ cells were collected using the Possel program and used as smooth muscle/perivascular cells as previously described. Endothelial cells were then isolated from the remaining fraction using CD31 MicroBeads (Miltenyi Biotec) and the Possel program.

Isolation of pulmonary neuroendocrine cells (PNECs)

To enrich epithelial cells, lung cell suspensions were incubated with PE/Cy7-conjugated anti-CD326 (EpCAM) antibody (BioLegend) along with CD31 and CD45 MicroBeads (Miltenyi Biotec). CD31⁺ and CD45⁺ cells were then removed using the Depletes program on the autoMACS Pro Separator.

The remaining cells were subsequently incubated with anti-PE MicroBeads UltraPure (Miltenyi Biotec), and EpCAM⁺ epithelial cells were enriched using the Posseld2 program. PNECs were identified using GAD67-GFP knock-in mice, in which neuroendocrine cells express GFP. After staining with 7-AAD to exclude dead cells, EpCAM⁺ GFP⁺ cells were sorted using FACSAria Fusion with FACSDiva (v9.0.1) (BD Biosciences, Franklin Lakes, NJ, USA).

Isolation of ILC2s and Th2 cells

For isolating lymphocyte populations, lung cell suspensions were stained with biotinylated lineage markers (CD11b, CD11c, NK-1.1, F4/80, Gr-1, B220, CD19, and TER-119) and incubated with Streptavidin MicroBeads (Miltenyi Biotec). Lineage-positive cells were then depleted using the Depletes program on an autoMACS Pro Separator. The remaining cells were stained with antibodies against CD3ε, CD4, CD25, CD45.2, T1/ST2, TCRβ, Thy1.2, CD127, and KLRG1. ILC2s were identified and isolated as Lin⁻ CD3ε⁻ CD4⁻ TCRβ⁻ Thy1.2⁺ CD127⁺ KLRG1⁺ cells as previously described, while Th2 cells were defined as CD3ε⁺ CD4⁺ TCRβ⁺ T1/ST2⁺ cells. Cell sorting was performed using a FACSAria Fusion with FACSDiva. The gating strategies are shown in Supplementary Figure S1, and the antibodies used for flow cytometry are listed in Supplementary Table S1.

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA extraction and RT-PCR were performed as described previously. Briefly, RT-qPCR cycling conditions were as follows: an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of denaturation (95 °C for 10 s) and annealing/extension (60 °C for 30 s). Fold changes in gene expression were determined using the standard curve method. The primers used for RT-PCR are listed in Supplementary Table S2.

Periodic acid-Schiff (PAS) staining

PAS staining was performed as previously described. PAS-stained sections were observed under a light microscope (CX43; EVIDENT, Tokyo, Japan).

Immunohistochemistry

Immunohistochemistry were perfomed as previously described. Primary antibodies used for immunohistochemistry are listed in Supplementary Table S3. For selected paraffin-embedded tissue sections, tyramide signal amplification (TSA)–based immunofluorescence staining was performed. Sections (6-µm thick) were deparaffinized in xylene and rehydrated in decreasing ethanol series. Antigen retrieval was performed by heating the sections in citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched by incubation with hydrogen peroxide, followed by blocking of non-specific binding using Blocking One (Nacalai Tesque). Sections were incubated with primary antibodies raised in rabbit, followed by horseradish peroxidase (HRP)–conjugated anti-rabbit IgG secondary antibodies. Immunoreactivity was visualized using a tyramide signal amplification (TSA) system with tyramide-Cy3 and tyramide-Cy5 (AAT Bioquest, Inc., Pleasanton, CA, USA). For sequential multiplex staining, bound antibodies were removed by heating the sections in citrate buffer (pH 6.0), and cycles of primary antibody incubation, HRP-conjugated secondary antibody incubation, and tyramide fluorophore labeling were repeated. Nuclei were counterstained with DAPI. All immunofluorescence images were captured using the ZEISS LSM 900 confocal laser scanning microscope.

Enzyme-linked immunosorbent assay (ELISA)

ELISA was performed as described previously. Protein concentrations in lung homogenates, plasma, and cell culture supernatants were measured using specific ELISA kits for GDNF (Invitrogen, Carlsbad, CA, USA), as well as for IL-5 and IL-13 (Thermo Fisher Scientific, Waltham, MA, USA). All procedures were performed according to the manufacturer’s instructions. ELISA for OVA-IgE was performed as previously described.

Assessment of GDNF-producing cells by flow cytometry

Lung cell suspensions were processed as described above. Live/dead discrimination was performed using Zombie Aqua viability dye. Cells were blocked with anti-mouse CD16/32 (Fc) antibody and anti-mouse IgG Fab fragment for 10 minutes at 4°C. Surface staining was performed with CD11c and Siglec-F (biotin) antibodies. After washing, cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (Invitrogen), and intracellular GDNF (Santa Cruz Biotechnology, 1:50) was stained at 4°C for 60 minutes. Secondary detection was performed with Alexa Fluor 488-conjugated anti-mouse IgG for GDNF and Alexa Fluor 555-conjugated Streptavidin for Siglec-F. Cells were filtered through a 70-µm strainer and analyzed on the FACSAria Fusion with FACSDiva. Eosinophils were identified as Siglec-F⁺ and CD11c⁻ cells, while macrophages were identified as Siglec-F⁺ and CD11c⁺ cells. The gating strategy is shown in Figure 3A.

Quantification of inflammatory cells in the bronchoalveolar lavage fluid (BALF)

BALF was collected 3 days after the final OVA challenge (day 27), following a previously described method.

Protocol for treatment with pralsetinib

Mice with asthma were treated with either pralsetinib (10 mg/kg, BMS-927711; Selleck Chemicals, Houston, TX, USA) or a vehicle of 5% dimethylsulfoxide and 95% corn oil via an oral sonde (Natsume Seisakusho, Tokyo, Japan) once daily between days 18 and 27. The administered volume was 200 μL, and the vehicle-treated group received an equivalent volume. The administration schedules are summarized in Figure 4A. The dose administered was determined based on a previous study.

Statistical analyses

Data are expressed as the mean ± standard deviation (SD) from multiple independent experiments (indicated by n values). Significant differences were determined using an unpaired t-test for normal distribution and either the Mann–Whitney U test or one-way analysis of variance (ANOVA) and Tukey’s post hoc-test for all other cases. Statistical analyses were performed using Prism 6 (GraphPad Software, San Diego, CA, USA). Statistical significance was set at p < 0.05.