Mononuclear cell composition and activation in blood and mucosal tissue of eosinophilic esophagitis.

Introduction: Eosinophilic esophagitis (EoE) is a chronic, inflammatory, antigen-driven disease of the esophagus. Tissue EoE pathology has previously been extensively characterized by novel transcriptomics and proteomic platforms, however the majority of surface marker determination and screening has been performed in blood due to mucosal tissue size limitations. While eosinophils, CD4+ T cells, mast cells and natural killer (NK) T cells were previously investigated in the context of EoE, an accurate picture of the composition of peripheral blood mononuclear cells (PBMC) and their activation is missing. Methods: In this study, we aimed to comprehensively analyze the composition of peripheral blood mononuclear cells and their activation using surface marker measurements with multicolor flow cytometry simultaneously in both blood and mucosal tissue of patients with active EoE, inactive EoE, patients with gastroesophageal reflux disease (GERD) and controls. Moreover, we set out to validate our data in co-cultures of PBMC with human primary esophageal epithelial cells and in a novel inducible mouse model of eosinophilic esophagitis, characterized by extensive IL-33 secretion in the esophagus. Results: Our results indicate that specific PBMC populations are enriched, and that they alter their surface expression of activation markers in mucosal tissue of active EoE. In particular, we observed upregulation of the immunomodulatory molecule CD38 on CD4+ T cells and on myeloid cells in biopsies of active EoE. Moreover, we observed significant upregulation of PD-1 on CD4+ and myeloid cells, which was even more prominent after corticosteroid treatment. With co-culture experiments we could demonstrate that direct cell contact is needed for PD-1 upregulation on CD4+ T cells. Finally, we validated our findings of PD-1 and CD38 upregulation in an inducible mouse model of EoE. Discussion: Herein we show significant alterations in the PBMC activation profile of patients with active EoE in comparison to inactive EoE, GERD and controls, which could have potential implications for treatment. To our knowledge, this study is the first of its kind expanding the multi-color flow cytometry approach in different patient groups using in vitro and in vivo translational models.

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.

Allergies Come Clean: The Role of Detergents in Epithelial Barrier Dysfunction.

PURPOSE OF REVIEW: The prevalence and incidence of allergic disease have been rising in Westernized countries since the twentieth century. Increasingly, evidence suggests that damage to the epithelium initiates and shapes innate and adaptive immune responses to external antigens. The objective of this review is to examine the role of detergents as a potential risk factor for developing allergic disease. RECENT FINDINGS: Herein, we identify key sources of human detergent exposure. We summarize the evidence suggesting a possible role for detergents and related chemicals in initiating epithelial barrier dysfunction and allergic inflammation. We primarily focus on experimental models of atopic dermatitis, asthma, and eosinophilic esophagitis, which show compelling associations between allergic disease and detergent exposure. Mechanistic studies suggest that detergents disrupt epithelial barrier integrity through their effects on tight junction or adhesion molecules and promote inflammation through epithelial alarmin release. Environmental exposures that disrupt or damage the epithelium may account for the increasing rates of allergic disease in genetically susceptible individuals. Detergents and related chemical compounds represent possible modifiable risk factors for the development or exacerbation of atopy.

Eosinophils promote effector functions of lung group 2 innate lymphoid cells in allergic airway inflammation in mice.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are critical mediators of type 2 respiratory inflammation, releasing IL-5 and IL-13 and promoting the pulmonary eosinophilia associated with allergen provocation. Although ILC2s have been shown to promote eosinophil activities, the role of eosinophils in group 2 innate lymphoid cell (ILC2) responses is less well defined. OBJECTIVE: We sought to investigate the role of eosinophils in activation of ILC2s in models of allergic asthma and in vitro. METHODS: Inducible eosinophil-deficient mice were exposed to allergic respiratory inflammation models of asthma, such as ovalbumin or house dust mite challenge, or to innate models of type 2 airway inflammation, such as inhalation of IL-33. Eosinophil-specific IL-4/13-deficient mice were used to address the specific roles for eosinophil-derived cytokines. Direct cell interactions between ILC2s and eosinophils were assessed by in vitro culture experiments. RESULTS: Targeted depletion of eosinophils resulted in significant reductions of total and IL-5+ and IL-13+ lung ILC2s in all models of respiratory inflammation. This correlated with reductions in IL-13 levels and mucus in the airway. Eosinophil-derived IL-4/13 was necessary for both eosinophil and ILC2 accumulation in lung in allergen models. In vitro, eosinophils released soluble mediators that induced ILC2 proliferation and G protein-coupled receptor-dependent chemotaxis of ILC2s. Coculture of ILC2s and IL-33-activated eosinophils resulted in transcriptome changes in both ILC2s and eosinophils, suggesting potential novel reciprocal interactions. CONCLUSION: These studies demonstrate that eosinophils play a reciprocal role in ILC2 effector functions as part of both adaptive and innate type 2 pulmonary inflammatory events.

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.

The role of biologics in pediatric food allergy and eosinophilic gastrointestinal disorders.

Continuing insight into the molecular mechanisms of atopic disorders has enabled the development of biologics to precisely target these diseases. Food allergy (FA) and eosinophilic gastrointestinal disorders (EGIDs) are driven by similar inflammatory molecular mechanisms and exist along the same atopic disease spectrum. Therefore, many of the same biologics are being investigated to target key drivers of mechanisms shared across the disease states. The enormous potential of biologics for the treatment of FA and EGIDs is highlighted by the significant increases in the number of ongoing clinical trials (more than 30) evaluating their use in these disease states, as well as by the recent US Food and Drug Administration approval of dupilumab for the treatment of eosinophilic esophagitis. Here we discuss past and current research into the use of biologics in FA and EGIDs and their potential role in improving treatment options in the future, with the need to have biologics widely clinically available.

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.

Immunologic and pathologic characterization of a novel swine biomedical research model for eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic allergy-mediated condition with an increasing incidence in both children and adults. Despite EoE's strong impact on human health and welfare, there is a large unmet need for treatments with only one recently FDA-approved medication for EoE. The goal of this study was to establish swine as a relevant large animal model for translational biomedical research in EoE with the potential to facilitate development of therapeutics. We recently showed that after intraperitoneal sensitization and oral challenge with the food allergen hen egg white protein (HEWP), swine develop esophageal eosinophilia-a hallmark of human EoE. Herein, we used a similar sensitization and challenge treatment and evaluated immunological and pathological markers associated with human EoE. Our data demonstrate that the incorporated sensitization and challenge treatment induces (i) a systemic T-helper 2 and IgE response, (ii) a local expression of eotaxin-1 and other allergy-related immune markers, (iii) esophageal eosinophilia (>15 eosinophils/0.24 mm2), and (iv) esophageal endoscopic findings including linear furrows and white exudates. Thereby, we demonstrate that our sensitization and oral challenge protocol not only induces the underlying immune markers but also the micro- and macro-pathological hallmarks of human EoE. This swine model for EoE represents a novel relevant large animal model that can drive translational biomedical research to develop urgently needed treatment strategies for EoE.

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.

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.

Gastrointestinal Eosinophil Responses in a Longitudinal, Randomized Trial of Peanut Oral Immunotherapy.

BACKGROUND & AIMS: Gastrointestinal side effects are common during oral immunotherapy (OIT) and eosinophilic esophagitis (EoE) is a potential complication. We aimed to characterize eosinophilic gastrointestinal responses to peanut OIT, in which peanut protein is given orally, with incremental increases in dose over time. METHODS: Twenty adults with IgE-mediated peanut allergy were randomly assigned to groups given peanut OIT (n = 15) or placebo (n = 5); 1 additional subject withdrew before randomization. Serial gastrointestinal biopsies were collected at baseline (n = 21, 0 weeks), following dose escalation (n = 10, 52 weeks), and during the maintenance phase (n = 11, 104 weeks). Endoscopic findings were characterized using the EoE endoscopic reference score. Biopsies were assessed for eosinophils per high-power field (eos/hpf) and other pathology features using EoE histologic scoring system scores. We performed immunohistochemical analyses of eosinophil peroxidase deposition, quantified using automated image analysis. RESULTS: At baseline, no subjects reported current gastrointestinal symptoms. However, 3 of the 21 subjects (14%) had esophageal peak eosinophil counts ≥15 eos/hpf and all subjects had dilated intercellular spaces (DIS). OIT induced or exacerbated esophageal eosinophilia (EE) at 52 weeks in most subjects (peak eosinophil counts >5 eos/hpf in 6 of 7 patients [86%]; peak eosinophil counts ≥15 eos/hpf in 4 of 7 patients [57%]). One subject met clinicopathologic criteria for EoE and withdrew; no significant changes in esophageal peak eosinophil counts were observed in the placebo group. EE in the OIT group corresponded with significant increases in EoE histologic scoring system scores and deposition of eosinophil peroxidase. In 4 of 6 participants (67%), OIT-induced EE and gastrointestinal eosinophilia resolved by the end of the maintenance phase. Gastrointestinal symptoms were not clearly associated with EE or gastrointestinal eosinophilia. CONCLUSIONS: In this pilot study, we found that peanut OIT-induced EE and gastrointestinal eosinophilia are usually transient and are not always associated with gastrointestinal symptoms. Clinicaltrials.gov no: NCT02103270.

Eosinophils in Eosinophilic Esophagitis: The Road to Fibrostenosis is Paved With Good Intentions.

Eosinophilic esophagitis (EoE) is an antigen-driven disease associated with epithelial barrier dysfunction and chronic type 2 inflammation. Eosinophils are the defining feature of EoE histopathology but relatively little is known about their role in disease onset and progression. Classically defined as destructive, end-stage effector cells, eosinophils (a resident leukocyte in most of the GI tract) are increasingly understood to play roles in local immunity, tissue homeostasis, remodeling, and repair. Indeed, asymptomatic esophageal eosinophilia is observed in IgE-mediated food allergy. Interestingly, EoE is a potential complication of oral immunotherapy (OIT) for food allergy. However, we recently found that patients with peanut allergy may have asymptomatic esophageal eosinophilia at baseline and that peanut OIT induces transient esophageal eosinophilia in most subjects. This is seemingly at odds with multiple studies which have shown that EoE disease severity correlates with tissue eosinophilia. Herein, we review the potential role of eosinophils in EoE at different stages of disease pathogenesis. Based on current literature we suggest the following: (1) eosinophils are recruited to the esophagus as a homeostatic response to epithelial barrier disruption; (2) eosinophils mediate barrier-protective activities including local antibody production, mucus production and epithelial turnover; and (3) when type 2 inflammation persists, eosinophils promote fibrosis.

Eosinophil-derived IL-13 promotes emphysema.

The inflammatory responses in chronic airway diseases leading to emphysema are not fully defined. We hypothesised that lung eosinophilia contributes to airspace enlargement in a mouse model and to emphysema in patients with chronic obstructive pulmonary disease (COPD).A transgenic mouse model of chronic type 2 pulmonary inflammation (I5/hE2) was used to examine eosinophil-dependent mechanisms leading to airspace enlargement. Human sputum samples were collected for translational studies examining eosinophilia and matrix metalloprotease (MMP)-12 levels in patients with chronic airways disease.Airspace enlargement was identified in I5/hE2 mice and was dependent on eosinophils. Examination of I5/hE2 bronchoalveolar lavage identified elevated MMP-12, a mediator of emphysema. We showed, in vitro, that eosinophil-derived interleukin (IL)-13 promoted alveolar macrophage MMP-12 production. Airspace enlargement in I5/hE2 mice was dependent on MMP-12 and eosinophil-derived IL-4/13. Consistent with this, MMP-12 was elevated in patients with sputum eosinophilia and computed tomography evidence of emphysema, and also negatively correlated with forced expiratory volume in 1 s.A mouse model of chronic type 2 pulmonary inflammation exhibited airspace enlargement dependent on MMP-12 and eosinophil-derived IL-4/13. In chronic airways disease patients, lung eosinophilia was associated with elevated MMP-12 levels, which was a predictor of emphysema. These findings suggest an underappreciated mechanism by which eosinophils contribute to the pathologies associated with asthma and COPD.

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.

Frontline Science: Eosinophil-deficient MBP-1 and EPX double-knockout mice link pulmonary remodeling and airway dysfunction with type 2 inflammation.

Eosinophils and the release of cationic granule proteins have long been implicated in the development of the type 2-induced pathologies linked with respiratory inflammation. Paradoxically, the ablation of the two genes encoding the most abundant of these granule proteins, major basic protein-1 (MBP-1) and eosinophil peroxidase (EPX), results in a near collapse of eosinophilopoiesis. The specificity of this lineage ablation and the magnitude of the induced eosinopenia provide a unique opportunity to clarify the importance of eosinophils in acute and chronic inflammatory settings, as well as to identify potential mechanism(s) of action linked with pulmonary eosinophils in those settings. Specifically, we examined these issues by assessing the induced immune responses and pathologies occurring in MBP-1-/-/EPX-/- mice after 1) ovalbumin sensitization/provocation in an acute allergen-challenge protocol, and 2) crossing MBP-1-/-/EPX-/- mice with a double-transgenic model of chronic type 2 inflammation (i.e., I5/hE2). Acute allergen challenge and constitutive cytokine/chemokine expression each induced the accumulation of pulmonary eosinophils in wild-type controls that was abolished in the absence of MBP-1 and EPX (i.e., MBP-1-/-/EPX-/- mice). The expression of MBP-1 and EPX was also required for induced lung expression of IL-4/IL-13 in each setting and, in turn, the induced pulmonary remodeling events and lung dysfunction. In summary, MBP-1-/-/EPX-/- mice provide yet another definitive example of the immunoregulatory role of pulmonary eosinophils. These results highlight the utility of this unique strain of eosinophil-deficient mice as part of in vivo model studies investigating the roles of eosinophils in health and disease settings.

Lung Pathologies in a Chronic Inflammation Mouse Model Are Independent of Eosinophil Degranulation.

RATIONALE: The release of eosinophil granule proteins in the lungs of patients with asthma has been dogmatically linked with lung remodeling and airway hyperresponsiveness. However, the demonstrated inability of established mouse models to display the eosinophil degranulation occurring in human subjects has prevented a definitive in vivo test of this hypothesis. OBJECTIVES: To demonstrate in vivo causative links between induced pulmonary histopathologies/lung dysfunction and eosinophil degranulation. METHODS: A transgenic mouse model of chronic T-helper cell type 2-driven inflammation overexpressing IL-5 from T cells and human eotaxin 2 in the lung (I5/hE2) was used to test the hypothesis that chronic histopathologies and the development of airway hyperresponsiveness occur as a consequence of extensive eosinophil degranulation in the lung parenchyma. MEASUREMENT AND MAIN RESULTS: Studies targeting specific inflammatory pathways in I5/hE2 mice surprisingly showed that eosinophil-dependent immunoregulative events and not the release of individual secondary granule proteins are the central contributors to T-helper cell type 2-induced pulmonary remodeling and lung dysfunction. Specifically, our studies highlighted a significant role for eosinophil-dependent IL-13 expression. In contrast, extensive degranulation leading to the release of major basic protein-1 or eosinophil peroxidase was not causatively linked to many of the induced pulmonary histopathologies. However, these studies did define a previously unappreciated link between the release of eosinophil peroxidase (but not major basic protein-1) and observed levels of induced airway mucin. CONCLUSIONS: These data suggest that improvements observed in patients with asthma responding to therapeutic strategies ablating eosinophils may occur as a consequence of targeting immunoregulatory mechanisms and not by simply eliminating the destructive activities of these purportedly end-stage effector cells.