Amanda B. Muir

Thymic stromal lymphopoietin–elicited basophil responses promote eosinophilic esophagitis

Eosinophilic esophagitis (EoE) is a food allergy–associated inflammatory disease characterized by esophageal eosinophilia. Current management strategies for EoE are nonspecific, and thus there is a need to identify specific immunological pathways that could be targeted to treat this disease. EoE is associated with polymorphisms in the gene that encodes thymic stromal lymphopoietin (TSLP), a cytokine that promotes allergic inflammation, but how TSLP might contribute to EoE disease pathogenesis has been unclear. Here, we describe a new mouse model of EoE-like disease that developed independently of IgE, but was dependent on TSLP and basophils, as targeting TSLP or basophils during the sensitization phase limited disease. Notably, therapeutic TSLP neutralization or basophil depletion also ameliorated established EoE-like disease. In human subjects with EoE, we observed elevated TSLP expression and exaggerated basophil responses in esophageal biopsies, and a gain-of-function TSLP polymorphism was associated with increased basophil responses in patients with EoE. Together, these data suggest that the TSLP-basophil axis contributes to the pathogenesis of EoE and could be therapeutically targeted to treat this disease.

Inflammation-associated microbiota in pediatric eosinophilic esophagitis

BackgroundEosinophilic esophagitis (EoE) is an allergic disorder characterized by eosinophil-predominant esophageal inflammation, which can be ameliorated by food antigen restriction. Though recent studies suggest that changes in dietary composition may alter the distal gut microbiome, little is currently known about the impact of a restricted diet upon microbial communities of the oral and esophageal microenvironments in the context of EoE. We hypothesize that the oral and esophageal microbiomes of EoE patients are distinct from non-EoE controls, that these differences correspond to changes in esophageal inflammation, and that targeted therapeutic dietary intervention may influence community structure. Using 16S rRNA gene sequencing, we characterized the bacterial composition of the oral and esophageal microenvironments using oral swabs and esophageal biopsies from 35 non-EoE pediatric controls and compared this cohort to samples from 33 pediatric EoE subjects studied in a longitudinal fashion before and after defined dietary changes.ResultsFirmicutes were more abundant in esophageal samples compared to oral. Proportions of bacterial communities were significantly different comparing all EoE esophageal microbiota to non-EoE controls, with enrichment of Proteobacteria, including Neisseria and Corynebacterium in the EoE cohort, and predominance of the Firmicutes in non-EoE control subjects. We detected a statistically significant difference between actively inflamed EoE biopsies and non-EoE controls. Overall, though targeted dietary intervention did not lead to significant differences in either oral or esophageal microbiota, reintroduction of highly allergenic foods led to enrichment in Ganulicatella and Campylobacter genera in the esophagus.ConclusionsIn conclusion, the esophageal microbiome in EoE is distinct from that of non-EoE controls, with maximal differences observed during active allergic inflammation.

Esophageal epithelial and mesenchymal cross-talk leads to features of epithelial to mesenchymal transition in vitro

BACKGROUND Esophageal fibrosis is a complication of eosinophilic esophagitis (EoE) which has been attributed to both subepithelial fibrosis and to epithelial to mesenchymal transition (EMT), a process by which epithelial cells acquire mesenchymal features. Common to both causes of EoE-fibrosis is the notion that granulocyte-derived TGF-β, induces myofibroblast differentiation of the target cell. To date, the role of esophageal epithelial cells as effector cells in esophageal fibrosis has never been explored. Herein, we investigated consequences of cross-talk between esophageal epithelial cells and fibroblasts, and identified profibrotic cytokines which influence the development of EMT in vitro. METHODS AND RESULTS Stimulation of primary fetal esophageal fibroblasts (FEF3) with conditioned media (CEM) from esophageal epithelial cells (EPC2-hTERT), primed FEF3 cells to secrete IL-1β and TNFα, but not TGFβ. To determine whether these cytokines signaled in a paracrine fashion to esophageal epithelial cells, FEF3 cells were stimulated with CEM, followed by transfer of this fibroblast conditioned media (FCM) to EPC2-hTERT cells. Epithelial FCM stimulation increased expression of mesenchymal markers and reduced E-cadherin expression, features of EMT which were TNFα and IL-1β-dependent. Using organotypic culture models, primary EoE epithelial cells exhibited features of EMT compared to non-EoE cells, corresponding to patterns of EMT in native biopsies. CONCLUSIONS Esophageal epithelial cell and fibroblast cross-talk contributes to esophageal fibrosis. Our results suggest that features of EMT can develop independent of TGF-β and granulocytes, which may have important implications in treatment of EoE.

Esophageal epithelial cells acquire functional characteristics of activated myofibroblasts after undergoing an epithelial to mesenchymal transition

BACKGROUND AND AIMS Eosinophilic esophagitis (EoE) is an allergic inflammatory disease that leads to esophageal fibrosis and stricture. We have recently shown that in EoE, esophageal epithelial cells undergo an epithelial to mesenchymal transition (EMT), characterized by gain of mesenchymal markers and loss of epithelial gene expression. Whether epithelial cells exposed to profibrotic cytokines can also acquire the functional characteristics of activated myofibroblasts, including migration, contraction, and extracellular matrix deposition, is relevant to our understanding and treatment of EoE-associated fibrogenesis. In the current study, we characterize cell migration, contraction, and collagen production by esophageal epithelial cells that have undergone cytokine-induced EMT in vitro. METHODS AND RESULTS Stimulation of human non-transformed immortalized esophageal epithelial cells (EPC2-hTERT) with profibrotic cytokines TNFα, TGFβ, and IL1β for three weeks led to acquisition of mesenchymal αSMA and vimentin, and loss of epithelial E-cadherin expression. Upon removal of the profibrotic stimulus, epithelial characteristics were partially rescued. TGFβ stimulation had a robust effect upon epithelial collagen production. Surprisingly, TNFα stimulation had the most potent effect upon cell migration and contraction, exceeding the effects of the prototypical profibrotic cytokine TGFβ. IL1β stimulation alone had minimal effect upon esophageal epithelial migration, contraction, and collagen production. CONCLUSIONS Esophageal epithelial cells that have undergone EMT acquire functional characteristics of activated myofibroblasts in vitro. Profibrotic cytokines exert differential effects upon esophageal epithelial cells, underscoring complexities of fibrogenesis in EoE, and implicating esophageal epithelial cells as effector cells in EoE-associated fibrogenesis.

Preferential Secretion of Thymic Stromal Lymphopoietin (TSLP) by Terminally Differentiated Esophageal Epithelial Cells: Relevance to Eosinophilic Esophagitis (EoE).

Eosinophilic esophagitis (EoE) is a chronic Th2 and food antigen-mediated disease characterized by esophageal eosinophilic infiltration. Thymic stromal lymphopoetin (TSLP), an epithelial derived cytokine which bridges innate and Th2-type adaptive immune responses in other allergic conditions, is overexpressed in esophageal biopsies of EoE subjects. However, the triggers of TSLP expression in the esophageal epithelium are unknown. The objective of the current study was to characterize TSLP expression in human esophageal epithelium in EoE in vivo and to determine the role of food antigens upon epithelial TSLP expression in vitro. Using immunohistochemistry (IHC), we localized TSLP in esophageal biopsies of active EoE (≥15 eos/hpf), inactive EoE (<15 eos/hpf) and non-EoE control subjects, and found that TSLP expression was restricted to the differentiated suprabasal layer of the epithelium in actively inflamed EoE biopsies. Consistent with these results in vivo, inducible TSLP protein secretion was higher in CaCl2 differentiated telomerase-immortalized esophageal epithelial cells (EPC2-hTERT) compared to undifferentiated cells of the basal phenotype, following stimulation with the TLR3 ligand poly(I:C). To determine whether food antigens could directly induce epithelial TSLP secretion, differentiated and undifferentiated primary esophageal epithelial cells from EoE and non-EoE subjects were challenged with food antigens clinically relevant to EoE: Chicken egg ovalbumin (OVA), wheat, and milk proteins beta-lactoglobulin (blg) and beta-casein. Food antigens failed to induce TSLP secretion by undifferentiated cells; in contrast, only OVA induced TSLP secretion in differentiated epithelial cells from both EoE and control cell lines, an effect abolished by budesonide and NF-κb inhibition. Together, our study shows that specific food antigens can trigger innate immune mediated esophageal TSLP secretion, suggesting that esophageal epithelial cells at the barrier surface may play a significant role in the pathogenesis of EoE by regulating TSLP expression.

Eosinophilic Esophagitis-associated Chemical and Mechanical Microenvironment Shapes Esophageal Fibroblast Behavior

Objectives: Eosinophilic esophagitis (EoE) is an immune-mediated allergic disease characterized by progressive esophageal dysmotility and fibrotic stricture associated with chronic esophageal fibroblast activation. It remains unknown how esophageal fibroblasts respond to EoE-relevant matrix stiffness or inflammatory cytokines. Methods: Immunofluorescence was used to evaluate &agr;-smooth muscle actin (&agr;-SMA) expression in endoscopic esophageal biopsies. Primary esophageal fibroblasts from adult and pediatric patients with or without EoE were exposed to transforming growth factor (TGF)&bgr; to determine gene expression, collagen-matrix contractility, and cytoskeletal organization. The influence of matrix stiffness upon fibroblast behavior was assessed on the engineered surface of polyacrylamide gels with varying stiffness. Fibroblast traction forces were measured using microfabricated-post-array-detectors. Results: EoE esophageal fibroblasts had enhanced &agr;-SMA expression. TGF&bgr; not only stimulated enhanced fibroblast-specific gene expression but also promoted fibroblast-mediated collagen-matrix contraction, despite disease state or age of patients as the origin of cells. Unlike conventional monolayer cell, culture conditions using plastic surface (1 GPa) that activates fibroblasts constitutively, our engineered platforms recapitulating physiologically relevant stiffness (1–20 kPa) revealed that matrix stiffness defines the extent of &agr;-SMA expression, intracellular collagen fibril organization, SMAD3 phosphorylation, and fibroblast traction force. Conclusions: Matrix stiffness may critically influence TGF&bgr;-mediated gene expression and functions of esophageal fibroblasts ex vivo independent of age and disease conditions. These findings provide a novel insight into the pathogenesis of fibrostenotic disease in EoE.

Microbiome and its impact on gastrointestinal atopy.

The prevalence of allergic conditions has continuously increased in the last few decades in Westernized countries. A dysbiotic gut microbiome may play an important role in the development of allergic diseases. Genetic, environmental, and dietary factors may alter the commensal microbiota leading to inflammatory dysregulation of homeostasis. Murine and human studies have begun to elucidate the role of the microbiota in the pathogenesis of atopic diseases including asthma, atopic dermatitis, and food allergies. However, the role of the microbiome in most eosinophilic gastrointestinal diseases (EGIDs) is not yet known. This review provides an overview of what is currently known about the development of tolerance from both molecular and clinical standpoints. We also look at the gut‐specific microbiome and its role in atopic conditions with the hope of applying this knowledge to the understanding, prevention, and treatment of EGIDs, particularly EoE.

Influence of Age and Eosinophilic Esophagitis on Esophageal Distensibility in a Pediatric Cohort

Objectives:Sequelae of eosinophilic esophagitis (EoE) include food impaction and esophageal stricture. Duration of inflammation is a predicted risk factor; however, complications remain unpredictable. Studies using the functional lumen imaging probe (FLIP) have demonstrated decreased distensibility of the esophagus in adult patients with EoE. As the impact of inflammation on the developing esophagus is unknown, we investigated esophageal distensibility in a pediatric cohort to determine the effect of age, ongoing inflammation, and fibrotic features on distensibility.Methods:We conducted a prospective observational study at two tertiary pediatric institutions. Subjects underwent FLIP evaluation during endoscopy to determine distensibility of the esophagus. During stepwise distension, simultaneous intrabag pressure and 16 channels of cross-sectional areas were measured. The minimal diameter at maximal esophageal distention at an intrabag pressure of 40 mm Hg was identified. Distensibility was compared between EoE and non-EoE subjects and between clinical variables within the EoE cohort. Potential confounding variables were identified.Results:Forty-four non-EoE and 88 EoE subjects aged 3–18 years were evaluated. Age positively correlated with esophageal distensibility in the non-EoE cohort, but this trend was not observed in the EoE population. Subjects with EoE had reduced distensibility even after adjusting for age. Active inflammation (eosinophils >15 eos/high-power field), histological lamina propria fibrosis, and various features of a fibrotic phenotype (stricture, food impaction, circumferential rings on endoscopy) were associated with decreased distensibility within the EoE cohort. FLIP was safe, feasible, and well tolerated.Conclusions:These findings suggest that remodeling occurs in the pediatric EoE population, warranting early diagnosis and initiation of therapy prior to the onset of disease complications.

Role of Endoscopy in Diagnosis and Management of Pediatric Eosinophilic Esophagitis.

Eosinophilic esophagitis (EoE) is a chronic allergic (immune-mediated) disease that leads to esophageal dysfunction and feeding disorders in children. Foods, and possibly environmental triggers, cause an inflammatory response in the esophagus, leading to esophageal inflammation, eosinophilic infiltration, and esophageal dysmotility, which may progress to dysphagia, food impaction, and esophageal stricture. Endoscopy with biopsy and histologic evaluation is currently the only method to diagnose EoE. Once diagnosed with EoE, children undergo follow-up endoscopy after therapy initiation and adjustments to ensure remission. Furthermore, children with food impactions or strictures may require endoscopic intervention such as foreign body removal and/or esophageal dilation.

Autophagy mediates epithelial cytoprotection in eosinophilic oesophagitis

Objective The influence of eosinophilic oesophagitis (EoE)-associated inflammation upon oesophageal epithelial biology remains poorly understood. We investigated the functional role of autophagy in oesophageal epithelial cells (keratinocytes) exposed to the inflammatory EoE milieu. Design Functional consequences of genetic or pharmacological autophagy inhibition were assessed in endoscopic oesophageal biopsies, human oesophageal keratinocytes, single cell-derived ex vivo murine oesophageal organoids as well as a murine model recapitulating EoE-like inflammation and basal cell hyperplasia. Gene expression, morphological and functional characterisation of autophagy and oxidative stress were performed by transmission electron microscopy, immunostaining, immunoblotting, live cell imaging and flow cytometry. Results EoE-relevant inflammatory conditions promoted autophagy and basal cell hyperplasia in three independent murine EoE models and oesophageal organoids. Inhibition of autophagic flux via chloroquine treatment augmented basal cell hyperplasia in these model systems. Oesophageal keratinocytes stimulated with EoE-relevant cytokines, including tumour necrosis factor-α and interleukin-13 exhibited activation of autophagic flux in a reactive oxygen species-dependent manner. Autophagy inhibition via chloroquine treatment or depletion of Beclin-1 or ATG-7, augmented oxidative stress induced by EoE-relevant stimuli in murine EoE, oesophageal organoids and human oesophageal keratinocytes. Oesophageal epithelia of paediatric EoE patients with active inflammation displayed increased autophagic vesicle content compared with normal and EoE remission subjects. Functional flow cytometric analysis revealed autophagic flux in human oesophageal biopsies. Conclusions Our findings reveal for the first time that autophagy may function as a cytoprotective mechanism to maintain epithelial redox balance and homeostasis under EoE inflammation-associated stress, providing mechanistic insights into the role of autophagy in EoE pathogenesis.