ABSTRACT
Background: The airway epithelium plays a central role in the pathogenesis of chronic respiratory diseases such as asthma and chronic rhinosinusitis with nasal polyps (CRSwNP), but the mechanisms by which airway epithelial cells (EpCs) maintain inflammation are poorly understood. Objective: We hypothesized that transcriptomic assessment of sorted airway EpCs across the spectrum of differentiation would allow us to define mechanisms by which EpCs perpetuate airway inflammation. Methods: Ethmoid sinus EpCs from adult patients with CRS were sorted into 3 subsets, bulk RNA sequenced, and analyzed for differentially expressed genes and pathways. Single cell RNA-seq (scRNA-seq) datasets from eosinophilic and non-eosinophilic CRSwNP and bulk RNA-seq of EpCs from mild/moderate and severe asthma were assessed. Immunofluorescent staining and ex vivo functional analysis of sinus EpCs were used to validate our findings. Results: Analysis within and across purified EpC subsets revealed an enrichment in glycolytic programming in CRSwNP vs CRSsNP. Correlation analysis identified mammalian target of rapamycin complex 1 (mTORC1) as a potential regulator of the glycolytic program and identified EpC expression of cytokines and wound healing genes as potential sequelae. mTORC1 activity was upregulated in CRSwNP, and ex vivo inhibition demonstrated that mTOR is critical for EpC generation of CXCL8, IL-33, and CXCL2. Across patient samples, the degree of glycolytic activity was associated with T2 inflammation in CRSwNP, and with both T2 and non-T2 inflammation in severe asthma. Conclusions: Together, these findings highlight a metabolic axis required to support epithelial generation of cytokines critical to both chronic T2 and non-T2 inflammation in CRSwNP and asthma.
ABSTRACT
BACKGROUND: Chronic rhinosinusitis with nasal polyposis (CRSwNP) is a type 2 (T2) inflammatory disease associated with an increased number of airway basal cells (BCs). Recent studies have identified transcriptionally distinct BCs, but the molecular pathways that support or inhibit human BC proliferation and differentiation are largely unknown. OBJECTIVE: We sought to determine the role of T2 cytokines in regulating airway BCs. METHODS: Single-cell and bulk RNA sequencing of sinus and lung airway epithelial cells was analyzed. Human sinus BCs were stimulated with IL-4 and IL-13 in the presence and absence of inhibitors of IL-4R signaling. Confocal analysis of human sinus tissue and murine airway was performed. Murine BC subsets were sorted for RNA sequencing and functional assays. Fate labeling was performed in a murine model of tracheal injury and regeneration. RESULTS: Two subsets of BCs were found in human and murine respiratory mucosa distinguished by the expression of basal cell adhesion molecule (BCAM). BCAM expression identifies airway stem cells among P63+KRT5+NGFR+ BCs. In the sinonasal mucosa, BCAMhi BCs expressing TSLP, IL33, CCL26, and the canonical BC transcription factor TP63 are increased in patients with CRSwNP. In cultured BCs, IL-4/IL-13 increases the expression of BCAM and TP63 through an insulin receptor substrate-dependent signaling pathway that is increased in CRSwNP. CONCLUSIONS: These findings establish BCAM as a marker of airway stem cells among the BC pool and demonstrate that airway epithelial remodeling in T2 inflammation extends beyond goblet cell metaplasia to the support of a BC stem state poised to perpetuate inflammation.
