Background: Eosinophilic oesophagitis (EoE) is a food allergen-induced inflammatory disorder characterised by interleukin (IL)-13-mediated oesophageal inflammation and epithelial basal cell hyperplasia (BCH). The role of mitochondria in EoE pathogenesis remains elusive. Design: Prompted by single cell transcriptomics data, we interrogated the role of mitochondria in EoE pathobiology using patient biopsies, EoE-mouse models and oesophageal epithelial cells grown in monolayer and three-dimensional (3D) organoid cultures treated with EoE-relevant cytokines. 3D organoids and EoE-bearing mice were treated with omeprazole—a proton-pump inhibitor used as first-line EoE therapy. We performed CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference in mouse organoids to identify the key mitochondrial regulatory genes whose depletion may lead to BCH. We analysed mitochondrial membrane potential, mass and superoxide production by flow cytometry, cellular oxygen consumption by respirometry, mitochondrial structures and perturbation of cellular energy homeostasis by immunoblotting. Results : Mitochondrial dysfunction appeared to be a hallmark of EoE-related BCH where mitochondrial structural damage was associated with impaired oxidative respiratory capacity, elevation of mitochondrial superoxide and decreased adenosine triphosphate (ATP) production, as corroborated by activation of the adenosine monophosphate (AMP) -activated protein kinase and suppression of mammalian target-of-rapamycin signalling. Depletion of PGC1A, the master regulator of mitochondria biogenesis, recapitulated EoE-related BCH, suggesting that mitochondrial dysfunction drives BCH. Further, omeprazole alleviated mitochondrial damage and dysfunction in EoE-related BCH modelled in mice and patient-derived organoids. Conclusion: Mitochondrial dysfunction is tightly linked to perturbation of redox homeostasis in EoE-related BCH, which is promoted by IL-13 and reversible with omeprazole treatment.

Background Eosinophilic oesophagitis (EoE) is a chronic allergic disease characterised by oesophageal epithelial remodelling, barrier dysfunction and inflammation. The transcription factor forkhead box M1 (FOXM1) has been shown to be a key regulator of epithelial proliferation and inflammation in allergic asthma. Objective To investigate the role of FOXM1 in epithelial disruption in EoE and to evaluate the therapeutic potential of FOXM1 inhibition. Design FOXM1 expression was analysed in human oesophageal biopsies, patient-derived organoids and murine EoE models. Interleukin (IL)-13 stimulation was used to model EoE in vitro. Using FOXM1 inhibition via the small molecule Robert Costa Memorial drug-1 (RCM-1), small interfering RNA-mediated knockdown or FOXM1 overexpression, the roles of FOXM1 were assessed by histology, gene expression profiling, organoid formation rates, barrier integrity and proliferation assays. RNA sequencing and chromatin immunoprecipitation were performed to elucidate molecular mechanisms. Results FOXM1 was significantly upregulated in patients with active EoE and localised to the basal epithelium. IL-13 increased FOXM1 expression in vitro. FOXM1 inhibition restored differentiation markers, reduced basal cell hyperplasia and proliferation, and improved barrier function. In a murine model, RCM-1 reduced epithelial changes and eosinophil infiltration. Conversely, FOXM1 overexpression promoted basal cell hyperplasia and proliferation. Mechanistically, FOXM1 directly regulated cell cycle gene, cyclin B1, which was upregulated in EoE and downregulated on FOXM1 inhibition. RCM-1 reduced phosphorylated STAT6 under IL-13 stimulation. FOXM1 expression was driven in part by an IL-13-PI3K/AKT axis. Conclusion FOXM1 plays a pivotal role in epithelial disruption in EoE by driving proliferation and impairing differentiation. Targeting FOXM1 restores epithelial homeostasis, mitigates inflammation and offers a novel therapeutic approach for EoE.

In homeostatic conditions, the basal progenitor cells of the esophagus differentiate into a stratified squamous epithelium. However, in the setting of acid exposure or inflammation, there is a marked failure of basal cell differentiation, leading to basal cell hyperplasia. We have previously shown that lysyl oxidase (LOX), a collagen crosslinking enzyme, is upregulated in the setting of allergic inflammation of the esophagus; however, its role beyond collagen crosslinking is unknown. Herein, we propose a non-canonical epithelial-specific role of LOX in the maintenance of epithelial homeostasis using 3D organoid and murine models. We performed quantitative reverse transcriptase PCR, Western blot, histologic analysis, and RNA sequencing on immortalized non-transformed human esophageal epithelial cells (EPC2-hTERT) with short-hairpin RNA (shRNA) targeting LOX mRNA in both monolayer and 3D organoid culture. A novel murine model with a tamoxifen-induced Lox knockout specific to the stratified epithelium (K5CreER; Lox^fl/fl) was utilized to further define the role of epithelial LOX in vivo. We found that LOX knockdown decreased the proliferative capacity of the esophageal epithelial cells in monolayer culture, and dramatically reduced the organoid formation rate (OFR) in the shLOX organoids. LOX knockdown was associated with decreased expression of the differentiation markers filaggrin, loricrin, and involucrin, with RNA sequencing analysis revealing 1224 differentially expressed genes demonstrating downregulation of pathways involved in cell differentiation and epithelial development. Mice with Lox knockout in their stratified epithelium demonstrated increased basaloid content of their esophageal epithelium and decreased Ki-67 staining compared to the vehicle-treated mice, suggesting reduced differentiation and proliferation in the Lox-deficient epithelium in vivo. Our results demonstrate, both in vivo and in vitro, that LOX may regulate epithelial homeostasis in the esophagus through the modulation of epithelial proliferation and differentiation. Understanding the mechanisms of perturbation in epithelial proliferation and differentiation in an inflamed esophagus could lead to the development of novel treatments that could promote epithelial healing and restore homeostasis.