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Metabolic dysfunction mediated by HIF-1α contributes to epithelial differentiation defects in eosinophilic esophagitis. [HIF1A-KD]

ABSTRACT: Investigating the contributory role that epithelial cell metabolism plays in allergic inflammation is a key factor to understanding what influences dysfunction and the pathogenesis of the allergic disease eosinophilic esophagitis (EoE). Herein, we sought to define the role of HIF-1α-mediated metabolic dysfunction in esophageal epithelial differentiation processes and barrier function in EoE. In RNA-sequencing derived from EoE patient biopsies, we observed the expression pattern of key genes involved in mitochondrial metabolism/oxidative phosphorylation (OXPHOS) and glycolysis. Bioenergetics analysis using Seahorse was performed on EPC2-hTERT cells to decipher the metabolic processes involved in epithelial differentiation processes. In addition, air-liquid interface cultures were employed to delineate metabolic dependency mechanisms required for epithelial differentiation. Transcriptomic analysis identified an increase in genes associated with OXPHOS in patients with EoE. Bioenergetic analysis in vitro revealed thatdifferentiated epithelium was less reliant on OXPHOS compared withundifferentiated epithelium. Increased OXPHOS potential and reduced glycolytic capacity was mirrored in HIF1A-knockdown EPC2-hTERT cells which portray a significant absence of terminal markers of epithelial differentiation, including involucrin. Pharmacological glucose transport inhibition phenocopied this, while rescue of the HIF-1α-deficient phenotype using the pan-prolyl hydroxylase inhibitor DMOG resulted in restored expression of epithelial differentiation markers. An OXPHOS-dominated metabolic pattern in EoE patients, brought about largely by the absence of HIF-1α-mediated glycolysis, is linked with the deficit in esophageal epithelial differentiation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE261362 | GEO | 2025/03/31

REPOSITORIES: GEO

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Project description:Introduction Disruption of the epithelial barrier can induce eosinophilic esophagitis (EoE), a TH2-mediated food- and aeroallergen associated chronic inflammatory disease 1, 2. The expression of IL-20 subfamily cytokines, IL-19, IL-20 and IL-24, is increased in TH2-mediated diseases, yet their function in EoE is unknown. Methods Combining RNA-sequencing (RNA-seq) and proteome analysis, we have examined the effects of the IL-20 subfamily on esophageal epithelial cells using patient-derived organoids and an experimental EoE mouse model. Results When stimulated with IL-20 subfamily cytokines patient-derived esophageal organoids showed decreased expression of Filaggrin (FLG) and Filaggrin 2 (FLG2) responsible for epithelial cornification. In agreement, EoE patients with active inflammation showed elevated IL-20 subfamily cytokines and decreased FLG and FLG2 expression. Topical corticosteroid treatment in EoE patients restored filaggrin expression, while reducing IL-20 subfamily cytokines. Abolishment of IL-20 subfamily signalling in Il20R2-/- animals attenuated experimental EoE in an OVA-induced mouse model. In the esophageal keratinocyte cell line, KYSE-180, we identified IL-20 subfamily-induced STAT3 and MAPK (ERK1/2) pathway activation. However, Stat3fl/flKrt5cre mice with an epithelium specific deletion of Stat3 developed exacerbated experimental EoE with a pronounced decrease of Flg2. In line, combined stimulation of patient-derived esophageal organoids with IL-20 subfamily cytokines and pharmacological inhibition of STAT3 resulted in aggravated decrease of FLG and FLG2. In addition, pharmacological STAT3 inhibition resulted in reduced transepithelial electrical resistance (TEER) and increased macromolecular flux in patient-derived air liquid interface (ALI) cultures. Whereas, inhibition of the MAPK (ERK1/2) pathway hampered IL-20 subfamily induced TEER reduction and paracellular flux increment. Finally, MAPK (ERK1/2) inhibitor treatment reduced infiltrating CD45+ immune cells in the esophagus and alleviated experimental EoE in mice. Conclusion We discovered a novel regulatory function of the IL-20 subfamily for epithelial barrier integrity. Aberrant IL-20 subfamily signaling disturbs the epithelial barrier function, while abrogation of IL-20 subfamily and ERK1/2 signaling restores epithelial integrity and attenuates experimental EoE. Therefore, we propose that targeting the IL-20 subfamily pathway could present a novel therapeutic strategy for EoE treatment.

Project description:RNA sequencing data of SPINK7 gene silencing (SPINK7) as compared to Non silencing control (NSC) treatment in EPC2 cells differentiated in ALI culture for 14 days and RNA sequencing data of NSC cells before differentiation. We show that a serine protease inhibitor SPINK7, is part of the differentiation program of human esophageal squamous epithelium and that SPINK7 depletion occurs in a human allergic, inflammatory, esophageal condition termed eosinophilic esophagitis (EoE). Herein, we employed experimental manipulation strategies designed to reduce SPINK7 in vitro in order to assess the transcriptional changes that are associates with the loss of SPINK7. A whole-transcriptome sequencing analysis of EPC2 cells after ALI differentiation revealed 270 genes that were differentially expressed in the SPINK7-deficient cells compared to NSC-treated cells (p < 0.05, fold change >2, RPKM >1). The modified genes were enriched for those involved in epidermal differentiation, inflammation and skin physiology including known differentiation markers such as FLG, FLG2, LOR, and keratins. These data suggest that loss of SPINK7 promote an un-differentiated phenotype. We further analyzed the transcription profile of differentiated cells as compared to un-differentiated cells. This analysis revealed 3225 differentially expressed genes (p < 0.05, fold change >2, RPKM >1). The majority of the genes modified by SPINK7 deficiency (77%) were also modulated during epithelial cell differentiation. Importantly, the genes that were most overexpressed during differentiation were downregulated after SPINK7 silencing, indicating that loss of SPINK7 promotes transcriptional changes that result in an undifferentiated epithelial phenotype. We propose that loss of SPINK7 is a key checkpoint in regulating mucosal differentiation, barrier function, and inflammatory esophageal responses. mRNA profile of SPINK7 silenced cells and control cells before and after differentiation in the ALI differentiation

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Metabolic dysfunction mediated by HIF-1α contributes to epithelial differentiation defects in eosinophilic esophagitis. [HIF1A-KD]

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