Increased epithelial mTORC1 activity in chronic rhinosinusitis with nasal polyps

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.

Increased glycolysis and cellular crosstalk in eosinophilic chronic rhinosinusitis with nasal polyps

“…To identify cellular functions associated with glycolytic reprogramming in CRS, we performed correlation analyses of the Mootha glycolysis module scores with gene sets in the Gene Ontology Biological Processes database (7655 gene sets) and Reactome database (1615 gene sets), followed by strict Bonferroni multiple testing correction (see Supplementary Table 1 for all significant results). We previously demonstrated using bulk RNA-seq that sinonasal basal EpC glycolysis is correlated with genes involved in wound healing ( 15 ) and, consistent with this, basal EpC glycolysis in the Wang dataset correlated with GOBP and Reactome gene sets related to cell motility ( Figure 3A ). These included genes encoding actin-related protein 2/3 complex ( ARPC2 ) ( 32 ), the Rho GTPase Cdc42 ( CDC42 ) ( 33 ), filamin A ( FNLA ) ( 34 ), midkine ( MDK ) ( 35 ), the scaffold protein NEDD9 ( NEDD9 ) ( 36 ), and other genes previously reported to control epithelial cell migration, metastasis, and invasion.…”

“…Our group and others have previously identified enhanced glycolysis in nasal epithelial cells (EpCs) in T2-high eCRSwNP ( 15 , 28 ). In that study, we performed unsupervised gene set enrichment analysis (GSEA) to demonstrate increased expression of the Hallmark glycolysis gene set in CRSwNP basal EpCs compared to CRSsNP basal EpCs, and we determined that expression of the Hallmark glycolysis gene set was tightly correlated with mTORC1 signaling in CRSwNP basal EpCs and with CD4 + Th2 cell infiltration in CRSwNP ( 15 ). To extend our metabolic profiling to additional pathways, cells, and disease subsets here, we assessed expression of the Mootha metabolic gene sets ( 17 , 29 ).…”

“…We previously identified that enhanced glycolysis in CRSwNP basal EpCs correlates with the expression of EpC wound healing genes ( 15 ). In this study, we sought to investigate if enhanced glycolysis was unique to EpCs within CRSwNP tissue.…”

“…Single cell RNA sequencing (scRNA-seq) FASTQ files of nasal mucosa from healthy controls (n=5), CRSsNP (n=5), neCRSwNP (n=5), and eCRSwNP (n=6) were downloaded from Genome Sequence Archive [HRA000772] ( 13 ) and processed as previously described ( 15 ). Clustering and sub-clustering reproduced the global structure reported by Wang et al.…”

Autophagy in chronic rhinosinusitis with or without nasal polyps

“…Epithelial glycolysis can be induced by Th2 and Th17 cytokines in vitro and is correlated with Th2 cytokine response in vivo . These findings imply that CRSwNP may exhibit autophagy deficiency, with mTORC1 playing a significant role in its pathogenesis, potentially linked to glycolytic activity ( Huang et al, 2023 ). Additionally, bulk and single cell RNA-seq data highlighted the importance of Migration Inhibitory Factor (MIF) and Transforming Growth Factor-Beta (TGF-β) pathways in mediating the effects of mitophagy in CRS, particularly emphasizing the role of MIF ( Zhou et al, 2023 ).…”