Solitary chemosensory cells are a primary epithelial source of IL-25 in patients with chronic rhinosinusitis with nasal polyps

Abstract

Background: IL-25 can function as an early signal for the respiratory type 2 response characteristic of allergic asthma and chronic rhinosinusitis with nasal polyps (CRSwNP). In the mouse gut, tuft cells are the epithelial source of IL-25. However, the source of human airway epithelial IL-25 has remained elusive.

Objective: In this study we sought to determine whether the solitary chemosensory cell (SCC) is the predominant source of IL-25 in the sinonasal epithelium.

Method: Flow cytometry and immunofluorescence for SCCs and IL-25 were used to interrogate polyp and turbinate tissue from patients with CRSwNP. Mucus was collected during acute inflammatory exacerbations from patients with CRSwNP or chronic rhinosinusitis without nasal polyps and IL-25 levels determined by using ELISA. Lastly, sinonasal epithelial cultures derived from polyp and turbinate tissue were stimulated with IL-13 and analyzed for SCC proliferation and IL-25 production.

Results: This study demonstrates that a discrete cell type, likely an SCC, characterized by expression of the taste-associated G protein gustducin and the intestinal tuft cell marker doublecortin-like kinase 1, is the predominant source of IL-25 in the human upper airway. Additionally, we show that patients with CRSwNP have increased numbers of SCCs in nasal polyp tissue and that in vitro IL-13 exposure both increased proliferation and induced apical secretion of IL-25 into the mucosal layer.

Conclusions: Inflammatory sinus polyps but not adjacent turbinate tissue show expansion of the SCC population, which is the source of epithelial IL-25.

Keywords: IL-13; IL-25; chronic rhinosinusitis with nasal polyps; doublecortin-like kinase 1; gustducin; solitary chemosensory cells; type 2 inflammation.

Fig. 1.. Gustducin-positive cells express DCAMKL-1 and IL-25 in differentiated human sinus epithelial cultures and tissue.

(A) Hi-resolution image depicting overlap in fluorescence between DCAMKL1 (green) and gustducin (red). (B) Co-labeling of gustducin-positive cells with IL-25. (C) Overlap between gustducin-positive (red) and cytoplasm-positive DCAMKL-1 cells (green) in human sinus polyp epithelium. Note scattered DCAMKL-1 positive nuclei in discrete cell population (white arrow). White bracket denotes edges of epithelium. (D) Co-labeling of gustducin/IL-25 positive cells in human sinus polyp epithelium. Nuclei labeled with DAPI depicted in blue, and DIC overlay for tissue on merged images. Scale bar 10 μm.

Fig. 2.. SCCs and IL-25 are increased in polyp tissue compared to turbinate tissue.

(A) IL-25/total protein (pg/mg) for matching polyp and turbinate samples from 15 CRSwNP patients demonstrates a significant increase (P = 0.0395) in IL-25 in polyp tissue (122 pg/mg) compared to inferior turbinate (60 pg/mg). (B) Significant increase in the percent of SCCs (GNAT⁺DCAMKL1⁺IL-25⁺/EpCAM⁺) in sinus polyps [39% (95% confidence interval 33–53%), 10 patients] versus control sinus tissue [8%, 995% confidence interval 0.6–24%) 8 patients] as determined by flow cytometry. (C) Percent of IL-25-positive epithelial cells that are DCAMKL1⁺/GNAT⁺ in polyp tissue (89%, 10 patients) versus control tissue (81%, 8 patients) as determined by flow cytometry. Shown are the individual data points (filled circles), the median (bar graph) +/− 95% confidence interval. (D,E) Representative gating strategy to determine percent of IL-25-positive epithelial cells that are DCAMKL1⁺/GNAT⁺ in polyp (D) and control (E) tissue.

Fig. 3.. IL-25-positive SCCs are enriched in polyp tissue.

Immunofluorescence of pathology-confirmed, (A) eosinophil-rich polyp, and (B) turbinate tissue from a patient with inflammatory sinus polyps with DCAMKL1 (cyan), IL-25 (green), and Gustducin (red). Turbinate tissue is devoid of DCAMKL1, IL-25 and gustducin-positive cells. DAPI-stained nuclei depicted in blue in merged images. Scale bar 10μm.

Fig. 4.. IL-25 secretion increases only in IL-13 treated Polyp Epithelial Cultures.

(A-C) IL-25 secretion in growth media +/− 50ng/ml IL-13. (A) IL-25 (pg/ml) secretion increases over time (days) for IL-13 treated CRSwNP epithelial cultures (filled black square) compared to untreated CRSwNP epithelial cultures (open squares) grown in submersion. IL-25 (pg/ml) after 14 days of treatment with (B) or without (C) 50ng/ml IL-13 in epithelial cultures derived from polyp tissue, middle turbinate or inferior turbinate grown in submersion. IL-25 (pg/ml) in (D) apical fluid, (E) basal media and (F) cell lysate of differentiated epithelial cells grown at an air-liquid interface derived from polyp or turbinate tissue that were untreated or treated with 50ng/ml IL-13 for 14 days. (G) Representative imaging of differentiated polyp cultures stained for gustducin and IL-25 +/−50ng/ml IL-13 for 14 days. DAPI staining of nuclei depicted in blue on merge image. Scale bar 10 microns.

Fig. 5.. IL-25 is apically secreted in culture and elevated in nasal secretions of patients with CRSwNP.

(A) Change in TEER (ohms-cm²) over time (days) for differentiated (mature ALI) or immature ALI polyp cultures +/− 50ng/ml IL-13. *= P<0.0005, ** = P<0.0001. mean +/−s.e.m. (B) Apical to basal transport of 20kDa FITC-Dextran (μg/ml) for differentiated (mature ALI) or immature ALI polyp cultures +/− 50ng/ml IL-13 for 14 days. (C) IL-25 (pg/ml) in apical secretions and basal media of undifferentiated polyp cultures (new ALI) +/− 50ng/ml IL-13 for 14 days, (## - below limit of detection). (D) IL-25 levels (pg/mg) in nasal secretions [CRSwNP (N=15), CRSsNP (n=12), control (N=7)] – shown are the median +/− 95% confidence interval plus all individual data points.