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The study aims to understand cell type-specific nitric oxide signaling mechanisms in sinonasal epithelial cells.

June 17, 2026Open Access

Live Imaging of Nitric Oxide Dynamics Reveals Cell Type-Specific NO Signaling in Air–Liquid Interface Cultures of Human Sinonasal Epithelial Cells

SHSakura HirokaneHiroshima University KKKeiichiro KiyoharaHiroshima University STSachio TakenoHiroshima University

Key Points

The study aims to understand cell type-specific nitric oxide signaling mechanisms in sinonasal epithelial cells.
Human sinonasal tissues were collected from 20 patients with CRSwNP and 20 controls.
Air–liquid interface cultures were developed to differentiate epithelial cells.
Nitric oxide synthase expressions and fluorescence signals were evaluated using immunostaining and quantitative RT-PCR methods.
CRSwNP tissues showed elevated iNOS expression and iNOS/eNOS ratios, with no significant change in eNOS.
Fluorescence signals for nitric oxide were significantly higher in ciliated versus non-ciliated cells (80.3 vs. 49.3).
Selective iNOS inhibition reduced nitric oxide signals in ciliated cells but did not completely abolish them.

Abstract

Background/Objectives: Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with epithelial remodeling, impaired mucociliary clearance, and altered nitric oxide (NO) metabolism. However, cell type-specific mechanisms underlying epithelial NO signaling remain poorly understood. This study investigated NO-related signaling in differentiated human sinonasal epithelial cells. Methods: Human sinonasal tissues were obtained from patients with CRSwNP (n = 20) and control subjects (n = 20). Air–liquid interface (ALI) cultures were established from donor-derived epithelial cells. Ciliated and non-ciliated cells were identified by immunostaining for acetylated α-tubulin and BCAM. Expression of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) was analyzed by quantitative RT-PCR. Intracellular NO-related fluorescence signals were evaluated using DAF-FM fluorescence imaging. Results: CRSwNP tissues exhibited significantly increased iNOS expression and elevated iNOS/eNOS ratios, whereas eNOS expression did not differ significantly from that in controls. ALI cultures reproduced differentiated sinonasal epithelium containing both ciliated and non-ciliated cell populations. DAF-FM fluorescence signals were significantly higher in ciliated cells than in non-ciliated cells (80.3 ± 25.3 vs. 49.3 ± 21.1). Non-selective NOS inhibition markedly reduced fluorescence signals in both cell types, whereas selective iNOS inhibition reduced but did not abolish signals in ciliated cells. Conclusions: NO-related signaling appears to differ among epithelial cell subtypes. Persistence of fluorescence signals after selective iNOS inhibition suggests a contribution of constitutive NOS activity in ciliated cells, whereas non-ciliated cells appear to rely more heavily on iNOS-dependent pathways. These findings support the hypothesis that altered epithelial NO signaling contributes to epithelial dysfunction and impaired mucociliary homeostasis in CRSwNP.

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