Epithelial overexpression of IL-33 induces eosinophilic esophagitis dependent on IL-13.

BACKGROUND: Eosinophilic esophagitis (EoE) is an increasingly common inflammatory condition of the esophagus; however, the underlying immunologic mechanisms remain poorly understood. The epithelium-derived cytokine IL-33 is associated with type 2 immune responses and elevated in esophageal biopsy specimens from patients with EoE. OBJECTIVE: We hypothesized that overexpression of IL-33 by the esophageal epithelium would promote the immunopathology of EoE. METHODS: We evaluated the functional consequences of esophageal epithelial overexpression of a secreted and active form of IL-33 in a novel transgenic mouse, EoE33. EoE33 mice were analyzed for clinical and immunologic phenotypes. Esophageal contractility was assessed. Epithelial cytokine responses were analyzed in three-dimensional organoids. EoE33 phenotypes were further characterized in ST2-/-, eosinophil-deficient, and IL-13-/- mice. Finally, EoE33 mice were treated with dexamethasone. RESULTS: EoE33 mice displayed ST2-dependent, EoE-like pathology and failed to thrive. Esophageal tissue remodeling and inflammation included basal zone hyperplasia, eosinophilia, mast cells, and TH2 cells. Marked increases in levels of type 2 cytokines, including IL-13, and molecules associated with immune responses and tissue remodeling were observed. Esophageal organoids suggested reactive epithelial changes. Genetic deletion of IL-13 in EoE33 mice abrogated pathologic changes in vivo. EoE33 mice were responsive to steroids. CONCLUSIONS: IL-33 overexpression by the esophageal epithelium generated immunopathology and clinical phenotypes resembling human EoE. IL-33 may play a pivotal role in the etiology of EoE by activating the IL-13 pathway. EoE33 mice are a robust experimental platform for mechanistic investigation and translational discovery.

IL-13-induced STAT3-dependent signaling networks regulate esophageal epithelial proliferation in eosinophilic esophagitis.

BACKGROUND: Basal zone hyperplasia (BZH) and dilated intercellular spaces (DISs) are thought to contribute to the clinical manifestations of eosinophilic esophagitis (EoE); however, the molecular pathways that drive BZH remain largely unexplored. OBJECTIVE: We sought to define the role of IL-13-induced transcriptional programs in esophageal epithelial proliferation in EoE. METHODS: We performed RNA sequencing, bioinformatics, Western blot, reverse transcriptase quantitative PCR, and histologic analyses on esophageal biopsies from healthy control and patients with EoE, primary esophageal cells derived from patients with EoE, and IL-13-stimulated esophageal epithelial keratinocytes grown at the air-liquid interface (EPC2-ALI). Genetic (shRNA) and pharmacologic (proteolysis-targeting chimera degrader) approaches and in vivo model of IL-13-induced esophageal epithelial remodeling (Krt5-rtTA x tetO-IL-13Tg) were used to define the role of signal transducer and activator of transcription 3 (STAT3) and STAT6 and secreted frizzled-related protein 1 (SFRP1) in esophageal epithelial proliferation. RESULTS: RNA-sequencing analysis of esophageal biopsies (healthy control vs EoE) and EPC2-ALI revealed 82 common differentially expressed genes that were enriched for putative STAT3 target genes. In vitro and in vivo analyses revealed a link between IL-13-induced STAT3 and STAT6 phosphorylation, SFRP1 mRNA expression, and esophageal epithelial proliferation. In vitro studies showed that IL-13-induced esophageal epithelial proliferation was STAT3-dependent and regulated by the STAT3 target SFRP1. SFRP1 mRNA is increased in esophageal biopsies from patients with active EoE compared with healthy controls or patients in remission and identifies an esophageal suprabasal epithelial cell subpopulation that uniquely expressed the core EoE proinflammatory transcriptome genes (CCL26, ALOX15, CAPN14, ANO1, and TNFAIP6). CONCLUSIONS: These studies identify SFRP1 as a key regulator of IL-13-induced and STAT3-dependent esophageal proliferation and BZH in EoE and link SFRP1+ esophageal epithelial cells with the proinflammatory and epithelial remodeling response in EoE.

Food-Specific IgG4 Is Elevated Throughout the Upper Gastrointestinal Tract in Eosinophilic Esophagitis.

BACKGROUND: Food-specific immunoglobulin G4 (FS-IgG4) is associated with eosinophilic esophagitis (EoE); however, it is not clear whether production is limited to the esophagus. AIMS: To assess FS-IgG4 levels in the upper gastrointestinal tract and plasma and compare these with endoscopic disease severity, tissue eosinophil counts, and patient-reported symptoms. METHODS: We examined prospectively banked plasma, throat swabs, and upper gastrointestinal biopsies (esophagus, gastric antrum, and duodenum) from control (n = 15), active EoE (n = 24), and inactive EoE (n = 8) subjects undergoing upper endoscopy. Patient-reported symptoms were assessed using the EoE symptom activity index (EEsAI). Endoscopic findings were evaluated using the EoE endoscopic reference score (EREFS). Peak eosinophils per high-power field (eos/hpf) were assessed from esophageal biopsies. Biopsy homogenates and throat swabs were normalized for protein content and assessed for FS-IgG4 to milk, wheat, and egg. RESULTS: Median FS-IgG4 for milk and wheat was significantly increased in the plasma, throat swabs, esophagus, stomach, and duodenum of active EoE subjects compared to controls. No significant differences for milk- or wheat-IgG4 were observed between active and inactive EoE subjects. Among the gastrointestinal sites sampled, FS-IgG4 levels were highest in the esophagus. Esophageal FS-IgG4 for all foods correlated significantly across all sites sampled (r ≥ 0.59, p < 0.05). Among subjects with EoE, esophageal FS-IgG4 correlated significantly with peak eos/hpf (milk and wheat) and total EREFS (milk). EEsAI scores and esophageal FS-IgG4 levels did not correlate. CONCLUSIONS: Milk and wheat FS-IgG4 levels are elevated in plasma and throughout the upper gastrointestinal tract in EoE subjects and correlate with endoscopic findings and esophageal eosinophilia.

Detergent exposure induces epithelial barrier dysfunction and eosinophilic inflammation in the esophagus.

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic allergic disease associated with type 2 inflammation and epithelial barrier dysfunction. The etiology is unknown, however, genetic heritability studies suggest environmental factors play a key role in pathogenesis. Detergents, such as sodium dodecyl sulfate (SDS), are common ingredients in household products such as dish soap and toothpaste. We hypothesized detergent exposure decreases epithelial barrier function and induces esophageal inflammation. METHODS: Immortalized esophageal epithelial cells (EPC2) were cultured in air-liquid interface (ALI) and exposed to SDS. Barrier function/activity was assessed by transepithelial electrical resistance (TEER), FITC-dextran flux, and RT-PCR. Additionally, SDS-treated mouse esophageal organoids were evaluated for morphology. To investigate the effects of SDS in vivo, mice were treated with 0.5% SDS in drinking water for 14 days. Esophagi were assessed by gross morphology, histopathology, protein expression, and bulk RNA sequencing. RESULTS: When EPC2 cells were exposed to SDS (5 µg/ml) for 96 h, TEER decreased (p = 0.03), and FITC-dextran flux increased (p = 0.0002). mRNA expression of IL-33 increased 4.5-fold (p = 0.02) at 6 h and DSG1 decreased (p < 0.0001) by 72 h. Disrupted epithelial integrity was noted in SDS-treated esophageal organoids. When mice were exposed to SDS, they showed increased esophageal width, chemokine, and metalloprotease levels. Mice treated with SDS also showed increased IL-33 protein expression, basal zone hyperplasia, CD4+ cell infiltration, and esophageal eosinophilia. RNA sequencing revealed upregulation of immune response pathway genes. CONCLUSION: Exposure to SDS decreases esophageal barrier integrity, stimulates IL-33 production, and promotes epithelial hyperplasia and tissue eosinophilia. Detergents may be a key environmental trigger in EoE pathogenesis.

Eosinophil Peroxidase Staining Enhances the Diagnostic Utility of the Cytosponge in Eosinophilic Esophagitis.

INTRODUCTION: We aimed to assess the diagnostic utility of eosinophil peroxidase (EPX) staining on Cytosponge (CS) samples in eosinophilic esophagitis (EoE). METHODS: Esophageal biopsy (BX) samples from adult subjects with EoE were assessed using peak eosinophils per high-power field (eos/hpf), EPX, and the EoE histologic scoring system. EPX staining and eos/hpf were compared (BX vs CS). RESULTS: CS EPX positivity correlated with eos/hpf (CS [ r = 0.82, P < 0.0001]; BX [ r = 0.65, P < 0.0001]) and EoE histologic scoring system (grade [ r = 0.62, P < 0.00001]; stage [ r = 0.61, P < 0.0001]). CS EPX identified subjects with active EoE (area under the curve = 0.86, P < 0.0001). DISCUSSION: The correlation of CS EPX with eosinophilic inflammation and histologic disease severity supports its diagnostic utility in EoE.

Refractory eosinophilic cystitis controlled with low-dose cyclosporine therapy: A case report.

Eosinophilic cystitis (EC) is a rare disease of the bladder with no clear inciting etiology, pathogenesis, or standard treatment. We present the case of a 78-year-old woman with a three-year history of refractory EC with symptoms characterized by urinary frequency, gross hematuria, dysuria, and suprapubic pain. Despite treatment with a silver nitrate instillation, antibiotics, alpha-1 blockers, antihistamines, antimuscarinics, beta-3 agonists, and intravesical steroid injections, her symptoms persisted. She was then trialed on systemic therapies including prednisone, montelukast, and cyclosporine. Upon follow-up after initiation of therapy with low-dose cyclosporine she had an excellent response, both symptomatically and anatomically via cystoscopy.

Diagnosis of Pediatric Non-Esophageal Eosinophilic Gastrointestinal Disorders by Eosinophil Peroxidase Immunohistochemistry.

BACKGROUND: Diagnosis of non-esophageal eosinophilic gastrointestinal disorders requires quantification of tissue eosinophils. Our objective was to evaluate eosinophil peroxidase (EPX) immunohistochemistry (IHC) as a method for histologic diagnosis of eosinophilic gastritis (EG) and eosinophilic duodenitis (EoD). METHODS: We performed a retrospective analysis of biopsies from pediatric EG/EoD cases and controls. Subjects with EG or EoD had ≥30 eosinophils per high power field (eos/hpf) in ≥5 hpf in the stomach and/or ≥3 hpf in the duodenum, respectively. Controls had no histopathologic diagnosis recorded. Tissue eosinophil counts were assessed by hematoxylin & eosin stains. EPX stains were assessed using a unique histopathologic scoring system. Slides were digitized and EPX+ staining area/mm2 was quantified by image analysis. RESULTS: Twenty-six EG/EoD cases and 40 controls were analyzed. EPX scores and EPX/mm2 levels were markedly elevated in EG/EoD (p ≤ 0.0001). Eosinophil density (eos/mm2) correlated strongly with EPX scores and EPX/mm2 levels in the stomach (r ≥ 0.77) and moderately with EPX scores and EPX/mm2 levels in the duodenum (r ≥ 0.52); (p < 0.0001). EPX quantification identified EG/EoD subjects with high diagnostic accuracy (EPX score: AUC = 1 for EG and EoD; EPX/mm2: AUC = 0.98 (95%CI 0.96-1) for EG, AUC = 0.91 (95%CI 0.81-1) for EoD). CONCLUSION: EPX-based assessment of eosinophilic inflammation may facilitate automated histologic diagnosis.

Image Analysis of Eosinophil Peroxidase Immunohistochemistry for Diagnosis of Eosinophilic Esophagitis.

BACKGROUND: Diagnosis of eosinophilic esophagitis (EoE) requires manual quantification of tissue eosinophils. Eosinophil peroxidase (EPX) is an eosinophil-specific, cytoplasmic granule protein released during degranulation. AIMS: The objective of this study was to evaluate image analysis of EPX immunohistochemistry as an automated method for histologic diagnosis of EoE. METHODS: We performed a secondary analysis of prospectively collected esophageal biopsies obtained from adult subjects with EoE and controls. Tissue sections were stained with hematoxylin and eosin (H&E) and evaluated for peak eosinophils per high power field (eos/hpf). The same slides were de-stained and re-stained to detect EPX for direct comparison. Slides were digitized, and EPX staining area/mm2 was quantified using image analysis. Paired samples were compared for changes in EPX staining in treatment responders and non-responders. RESULTS: Thirty-eight EoE cases and 49 controls were analyzed. Among EoE subjects, matched post-treatment biopsies were available for 21 responders and 10 non-responders. Baseline EPX/mm2 was significantly increased in EoE subjects and decreased in treatment responders. EPX quantification correlated strongly with eos/hpf (r = 0.84, p < 0.0001) and identified EoE subjects with high diagnostic accuracy (AUC 0.95, p < 0.0001). The optimal diagnostic EPX-positive pixel/area threshold was 17,379 EPX/mm2. Several controls (5/49) with < 15 eos/hpf on H&E staining exceeded this cutoff. CONCLUSIONS: EPX/mm2 correlates strongly with eos/hpf, accurately identifies subjects with EoE, and decreases in treatment responders. Automated quantification of intact eosinophils and their degranulation products may enhance pathologic assessment. Future studies are needed to correlate EPX/mm2 with symptoms, endoscopic findings, and esophageal distensibility.

How to approach adult patients with asymptomatic esophageal eosinophilia.

As the awareness among gastroenterologists regarding endoscopic features suggesting eosinophilic esophagitis is increasing, individuals without symptoms of esophageal dysfunction are increasingly being found to have esophageal eosinophilia on biopsies performed during upper gastrointestinal endoscopies. However, the course of disease and the management of these asymptomatic individuals with esophageal eosinophilia remain elusive. In this review, we propose a definition of asymptomatic individuals with esophageal eosinophilia and discuss the prevalence, risk factors, and course of disease of this specific patient group. Furthermore, we have established a diagnostic and therapeutic pathway based on the most recent available data.

A Novel Allergen-Specific Immune Signature-Directed Approach to Dietary Elimination in Eosinophilic Esophagitis.

OBJECTIVES: Dietary elimination for treatment of eosinophilic esophagitis (EoE) is limited by lack of accuracy in current allergy tests. We aimed to develop an immunologic approach to identify dietary triggers and prospectively test allergen-specific immune signature-guided dietary elimination therapy. METHODS: In the first phase, we developed and assessed 2 methods for determining selected food triggers using samples from 24 adults with EoE: a CD4+ T-cell proliferation assay in peripheral blood and food-specific tissue IgG4 levels in esophageal biopsies. In the second phase, we clinically tested elimination diets created from these methods in a prospective cohort treated for 6 weeks (NCT02722148). Outcomes included peak eosinophil counts (eos/hpf), endoscopic findings (measured by the EoE Endoscopic Reference Score), and symptoms (measured by the EoE Symptom Activity Index). RESULTS: Parameters were optimized with a positive test on either assay, yielding agreements of 60%, 75%, 53%, 58%, and 53% between predicted and known triggers of peanut, egg, soy, wheat, and milk, respectively. In clinical testing, the mean number of foods eliminated based on the assays was 3.4, and 19 of 22 subjects were compliant with treatment. After treatment, median peak eosinophil counts decreased from 75 to 35 (P = 0.007); there were 4 histologic responders (21%). The EoE Endoscopic Reference Score and EoE Symptom Activity Index score also decreased after treatment (4.6 vs 3.0; P = 0.002; and 32.5 vs 25.0; P = 0.06, respectively). DISCUSSION: We successfully developed a new testing approach using CD4 T-cell proliferation and esophageal food-specific IgG4 levels, with promising accuracy rates. In clinical testing, this led to improvement in eosinophil counts, endoscopic severity, and symptoms of dysphagia, but a smaller than expected number of patients achieved histologic remission.

Development and Application of a Functional Human Esophageal Mucosa Explant Platform to Eosinophilic Esophagitis.

There is an increasing prevalence of esophageal diseases but intact human tissue platforms to study esophageal function, disease mechanisms, and the interactions between cell types in situ are lacking. To address this, we utilized full thickness human donor esophagi to create and validate the ex vivo function of mucosa and smooth muscle (n = 25). Explanted tissue was tested for contractile responses to carbachol and histamine. We then treated ex vivo human esophageal mucosa with a cytokine cocktail to closely mimic the Th2 and inflammatory milieu of eosinophilic esophagitis (EoE) and assessed alterations in smooth muscle and extracellular matrix function and stiffening. We found that full thickness human esophagus as well as the individual layers of circular and longitudinal muscularis propria developed tension in response to carbachol ex vivo and that mucosa demonstrated squamous cell differentiation. Treatment of mucosa with Th2 and fibrotic cytokines recapitulated the majority of the clinical Eosinophilic Esophagitis Diagnostic Profile (EDP) on fluidic transcriptional microarray. Transforming growth factor-beta-1 (TGFß1) increased gene expression of fibronectin, smooth muscle actin, and phospholamban (p < 0.001). The EoE cocktail also increased stiffness and decreased mucosal compliance, akin to the functional alterations in EoE (p = 0.001). This work establishes a new, transcriptionally intact and physiologically functional human platform to model esophageal tissue responses in EoE.

Baseline Gastrointestinal Eosinophilia Is Common in Oral Immunotherapy Subjects With IgE-Mediated Peanut Allergy.

Rationale: Oral immunotherapy (OIT) is an emerging treatment for food allergy. While desensitization is achieved in most subjects, many experience gastrointestinal symptoms and few develop eosinophilic gastrointestinal disease. It is unclear whether these subjects have subclinical gastrointestinal eosinophilia (GE) at baseline. We aimed to evaluate the presence of GE in subjects with food allergy before peanut OIT. Methods: We performed baseline esophagogastroduodenoscopies on 21 adults before undergoing peanut OIT. Subjects completed a detailed gastrointestinal symptom questionnaire. Endoscopic findings were assessed using the Eosinophilic Esophagitis (EoE) Endoscopic Reference Score (EREFS) and biopsies were obtained from the esophagus, gastric antrum, and duodenum. Esophageal biopsies were evaluated using the EoE Histologic Scoring System. Immunohistochemical staining for eosinophil peroxidase (EPX) was also performed. Hematoxylin and eosin and EPX stains of each biopsy were assessed for eosinophil density and EPX/mm2 was quantified using automated image analysis. Results: All subjects were asymptomatic. Pre-existing esophageal eosinophilia (>5 eosinophils per high-power field [eos/hpf]) was present in five participants (24%), three (14%) of whom had >15 eos/hpf associated with mild endoscopic findings (edema, linear furrowing, or rings; median EREFS = 0, IQR 0-0.25). Some subjects also demonstrated basal cell hyperplasia, dilated intercellular spaces, and lamina propria fibrosis. Increased eosinophils were noted in the gastric antrum (>12 eos/hpf) or duodenum (>26 eos/hpf) in 9 subjects (43%). EPX/mm2 correlated strongly with eosinophil counts (r = 0.71, p < 0.0001). Conclusions: Pre-existing GE is common in adults with IgE-mediated peanut allergy. Eosinophilic inflammation (EI) in these subjects may be accompanied by mild endoscopic and histologic findings. Longitudinal data collection during OIT is ongoing.

Minimally invasive biomarker studies in eosinophilic esophagitis: A systematic review.

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic, inflammatory disease of the esophagus that currently requires repeated endoscopic biopsies for diagnosis and monitoring because no reliable noninvasive markers have been identified. OBJECTIVE: To identify promising minimally invasive EoE biomarkers and remaining gaps in biomarker validation. METHODS: We performed a systematic review of EMBASE, Ovid MEDLINE, PubMed, and Web of Science from inception to June 6, 2017. Studies were included if patients met the 2007 consensus criteria for EoE diagnosis, a minimally invasive biomarker was assessed, and the study included at least 1 control for comparison. RESULTS: The search identified 2094 studies, with 234 reviewed at full-text level, and 49 included in the analysis (20 adult, 19 pediatric, 7 pediatric and adult, and 3 not stated). Most (26 of 49) were published after 2014. Thirty-five studies included healthy controls, 9 analyzed atopic controls, and 29 compared samples from patients with active and inactive EoE. Minimally invasive biomarkers were obtained from peripheral blood (n = 41 studies), sponge or string samples (n = 3), oral or throat swab secretions (n = 2), breath condensate (n = 2), stool (n = 2), and urine (n = 2). The most commonly reported biomarkers were peripheral blood eosinophils (n = 16), blood and string eosinophil granule proteins (n = 14), and eosinophil surface or intracellular markers (n = 12). EoE biomarkers distinguished active EoE from healthy controls in 23 studies, atopic controls in 2 studies, and inactive EoE controls in 20 studies. CONCLUSION: Several promising minimally invasive biomarkers for EoE have emerged; however, few are able to differentiate EoE from other atopic diseases.