Eosinophilic esophagitis (EoE) is a type 2 allergic disease characterized by esophageal inflammation and epithelial cell dysfunction. The acquired loss of the anti–serine protease of kazal type 7 (anti-SPINK7) in the squamous epithelium of the esophagus has a causal role in EoE pathogenesis. However, there is a limited understanding of the factors that regulate its expression and responsiveness to inflammatory stimuli. Herein, we have identified the transcription factor, ovo like transcriptional repressor 1 (OVOL1), as an esophageal selective gene product that regulates SPINK7 promoter activity. Overexpression of OVOL1 increased SPINK7 expression, whereas its depletion decreased SPINK7 expression, impaired epithelial barrier, and increased production of the proatopy cytokine thymic stromal lymphopoietin (TSLP). Stimulation with IL-13 abrogated the nuclear translocation of OVOL1 and promoted enhanced degradation of OVOL1 protein. This effect of IL-13 was dependent on the esophageal specific cysteine protease calpain-14 at least in part. Analysis of human esophageal biopsies demonstrated that the expression of esophageal OVOL1 correlated with SPINK7 transcript expression and was lost as a function of EoE disease activity. In summary, our study identifies key regulatory mechanisms in EoE pathogenesis, demonstrating that OVOL1 promotes SPINK7 transcription, whereas IL-13 suppresses this pathway in EoE.

BACKGROUND:Eosinophilic esophagitis (EoE) is an atopic disease driven in part by genetic susceptibility, but single-trait genome-wide association study (GWAS) has identified a limited number of genome-wide significant risk loci. OBJECTIVE:We sought to expand discovery of EoE genetic risk loci by leveraging shared genetic architecture with other atopic diseases and to develop a polygenic risk score (PRS) for EoE. METHODS:We performed a GWAS of 1,757 individuals with EoE and 14,467 population controls. We then applied multitrait analysis of GWAS (MTAG), integrating EoE with other atopic disease GWAS (UK Biobank; >450,000 subjects). Functional analyses were used to nominate candidate EoE risk genes. PRS models derived from MTAG were compared to PRS derived from the EoE-only GWAS. An interactive tool (EGIDExpress; https://egidexpress. RESEARCH:cchmc.org/GWAS/) was developed to enable dataset queries and visualization. RESULTS:The EoE-only GWAS identified 11 independent risk variants across 8 loci (P < 5 × 10-8), including 3 novel loci. MTAG identified 33 independent EoE risk variants across 24 loci, including 14 novel loci. Functional studies nominated 90 candidate EoE risk genes, including genes implicating mechanisms beyond type 2 immunity. A PRS derived from MTAG outperformed a PRS derived from the EoE-only GWAS (OR 11.57 [95% confidence interval, 6.90-19.40] for top vs bottom decile). CONCLUSION:Leveraging shared atopic disease genetics via MTAG substantially expands the landscape of EoE risk loci and improves EoE polygenic risk prediction, underscoring shared genetic mechanisms across atopic diseases. We further provide a public resource (EGIDExpress; https://egidexpress. RESEARCH:cchmc.org/GWAS/) to advance the field.

BACKGROUND:Immune tolerance in allergic diseases is associated with attenuation of TH2 responses by shifting of antigen-specific immunity toward TH1 and regulatory T-cell pathways, but current strategies incompletely induce durable regulatory immunity. OBJECTIVE:We determined whether combining an allergen-encoded messenger RNA (mRNA) lipid nanoparticle (LNP) vaccine with inhibition of the mechanistic target of rapamycin (mTOR) enhances regulatory T-cell responses. METHODS:Mice were immunized with an allergen-encoded mRNA-LNP vaccine alone or in combination with an mTOR inhibitor, followed by induction of a preclinical model of allergic asthma. Antigen-specific T-cell responses, eosinophil activation, airway hyperresponsiveness, mucus production, and markers of cytotoxicity were assessed. RESULTS:Immunization with allergen-encoded mRNA-LNP elicited TH1-associated and cytotoxic CD8+ T responses that counterbalanced TH2 immunity. Coadministration with an mTOR inhibitor shifted this profile by promoting generation of functional regulatory T cells and attenuating IFN-γ production and CD8+ T-cell responses. This combinatorial strategy preserved the antiallergic effects of mRNA-LNP immunization, reduced eosinophil activation markers, and limited vaccine-associated cytotoxicity. CONCLUSION:The ability of an mTOR inhibitor to profoundly modify mRNA-LNP therapy by inducing regulatory T cells presents a potential strategy to enhance regulatory immunity in the treatment of allergy and other inflammatory diseases.

BACKGROUND:Eosinophilic esophagitis (EoE) is a progressive fibrostenotic disease. Although proton pump inhibitors (PPIs) are a first-line EoE treatment due to their anti-inflammatory effects, their effects on remodeling/fibrosis-likely driven in part by TGF-β-remain uncertain. OBJECTIVES:To elucidate remodeling/fibrosis effects, this study evaluated whether PPIs impact the esophageal transcriptome of PPI-responsive EoE and counteract TGF-β‒induced fibrotic responses in human primary esophageal fibroblasts (HEFs). METHODS:Prospectively collected paired esophageal biopsies from patients with EoE pre‒/post‒PPI treatment were analyzed by RNA sequencing (RNA-seq). Histologic responsiveness to PPIs was defined as responders (<15 eosinophils/high-power field, n = 10) or nonresponders (≥15 eosinophils/high-power field, n = 9). The ability of PPIs (esomeprazole, omeprazole) to attenuate in vitro, TGF-β‒mediated remodeling/fibrosis in HEFs was analyzed by quantitative PCR, RNA-seq, Western blotting, immunofluorescence, cell migration assays, and reactive oxygen species measurements. RESULTS:In PPI responders, we identified 746 differentially expressed genes pre‒/post‒PPI treatment (≥2-fold change, P < .05), particularly those enriched in remodeling/fibrosis. In HEFs, TGF-β increased collagen I and α-smooth muscle actin expression via SMAD2/3 phosphorylation; however, PPIs attenuated these responses. RNA-seq revealed that PPIs reversed approximately 30% of TGF-β‒induced changes overlapping with fibrotic responses; 78 genes were concordantly modulated between patient biopsies and HEFs. Functional assays further confirmed that PPIs reduced TGF-β-induced collagen deposition, fibroblast motility, and reactive oxygen species production. CONCLUSIONS:PPIs modulate remodeling-/fibrosis-related gene expression in patients with EoE and inhibit TGF-β‒induced profibrotic responses in HEFs, supporting antifibrotic potential that may help limit fibrostenotic progression in EoE.

Eosinophils accumulate in different tissues in allergies, cancer, and infectious diseases, adopting distinct properties. Herein, we profiled murine esophageal eosinophils during allergic inflammation using single-cell sequencing, epigenomic mapping, and flow cytometry. Esophageal eosinophils displayed an altered chromatin accessibility profile compared to bone marrow eosinophils, with 761 epi-transcriptionally regulated genes enriched in inflammation, immunoregulation, bacterial sensing, angiogenesis, migration, and apoptosis. The local environment entrains the unique esophageal eosinophil immunophenotype, as suggested by eosinophil-esophageal epithelial co-cultures, esophageal eosinophil transcriptional similarities regardless of the upstream cytokines driving their esophageal localization, and transcription factor gene editing altering esophageal eosinophilia and the associated eosinophil and global esophageal transcriptomes. Finally, the epigenomic and transcriptomic properties of murine esophageal eosinophils are largely conserved in humans. Thus, our data indicate that tissue specialization of esophageal eosinophils is entrained by local environmental cues that induce genome-wide epigenetic reprogramming and regulated by discrete transcription factors and provide a public, epigenetic database of murine tissue eosinophils.

Atopic diseases, including eosinophilic esophagitis (EoE), are driven in part by genetic susceptibility. We performed a genome-wide association study (GWAS) of 1,757 EoE and 14,467 population controls, identifying 11 independent genetic risk variants spanning 8 EoE risk loci (p < 5x10 − 8 ), including 3 new loci. A multi-trait analysis of GWAS (MTAG) of EoE and other atopic diseases including over 450,000 subjects from the UK Biobank study identified 33 independent EoE genetic risk variants spanning 24 loci, including 14 novel loci. Functional studies nominated 90 EoE candidate genes, some involved in unexpected pathoetiology beyond type 2 immunity. A polygenic risk score derived from the MTAG replicated high risk of EoE compared with PRS derived from GWAS alone (OR 11.57 [6.90–19.40] in the top vs. bottom decile). An interactive tool (EGIDExpress) was developed to enable dataset queries and visualization. These findings offer expanded insight into EoE genetic risk and pathoetiology, underscore the genetic interplay of EoE with common atopic diseases, and provide a public resource that will advance the allergy field.

Allergic diseases have reached epidemic proportions globally, calling attention to the need for better treatment and preventive approaches. Herein, we developed allergen-encoding messenger RNA (mRNA)-lipid nanoparticle (LNP) strategies for both therapy and prevention of allergic responses. Immunization with allergen-encoded mRNA-LNPs modulated T cell differentiation, inhibiting the generation of T helper type 2 and type 17 cells upon allergen exposure in experimental asthma models induced by ovalbumin, and naturally occurring house dust mite (HDM) and the major HDM allergen Der p1. Allergen-specific mRNA-LNP treatment attenuated clinicopathology in both preventive and established allergy models, including reduction in eosinophilia, mucus production, and airway hypersensitivity, while enhancing production of allergen-specific IgG antibodies and maintaining low IgE levels. Additionally, allergen-specific mRNA-LNP vaccines in mice elicited a CD8+CD38+KLRG- T cell response as seen following SARS-CoV-2 mRNA vaccination in humans, underscoring a conserved immune mechanism across species, regardless of the mRNA-encoded protein. Notably, mRNA-LNP vaccination in combination with an mTOR inhibitor reduced the CD8+ T cell response without affecting the vaccine-induced anti-allergic effect in the preventive model of asthma. This technology renders allergen-specific mRNA-LNP therapy a promising approach for prevention and treatment of allergic diseases.

Background:Eosinophilic esophagitis is a chronic food induced allergic inflammatory disease of the esophagus. Eosinophil-depleting antibodies have not shown significant improvement in clinical symptoms, which has turned attention to mast cells. These cells also accumulate in the esophagus of EoE at levels that correlate with symptoms, demonstrate extensive activation including degranulation, correlate and remain elevated and poised for reactivation even in EoE patients in remission as defined by reduced eosinophil levels. Herein, we examine a mechanism of mast cell activation in EoE. Methods:Esophageal mast cell degranulation was quantitated by CD63 expression by flow cytometry. Mast cell degranulation activity of esophageal biopsies was examined employing a bioassay with human CD34 progenitor cell derived primary human mast cells. Bulk and single cell RNA sequencing data of the esophagus were analyzed for complement dysregulation and mast cell properties as a function of disease state. Human esophageal biopsies were stained for C3 and analyzed for C3a and C3 protein content. Primary human mast cells and fibroblasts were further interrogated by flow cytometry, immunostaining, and RNA sequencing. Results:Esophageal mast cells demonstrated increased CD63 expression, a measure of degranulation, in subjects with active compared with remission EoE. Esophageal biopsy lysates induced mast cell degranulation. Analysis of esophageal bulk RNA sequencing data demonstrated that the mast cell marker CPA3 strongly correlated with C3AR1 . Accordingly, the level of C3a receptor on esophageal mast cells was proportional with CD63 expression. C3AR1 expression was increased in the fibrostenotic endoscopic phenotype compared to normal appearing active EoE patients. Esophageal biopsies were notably enriched in the expression of multiple complement genes (including C3AR1 , C3 , CFB, C1QA, C1QB , and C1QC ) some of which were increased in active vs remission EoE ( C3AR1 , CFB, C1QA, C1QB , and C1QC ). Single cell RNA (scRNA) sequencing data revealed enrichment of C3 in fibroblasts at levels higher in EoE compared with non-EoE controls. The ratio of C3a/C3 protein levels in the esophagus was increased in EoE compared to control. Mast cells and fibroblasts have increased proximity in EoE compared with non-EoE control subjects. Additionally, C3a stimulated mast cells to express increased CD117 and release multiple EoE-relevant cytokines including fibroblast active mediators including granzyme B, which is enriched in esophageal mast cells. Conclusion:Taken together, our findings present evidence for the interplay of mast cells and fibroblasts via C3a in the pathogenesis of EoE.

The oesophagus has traditionally been viewed as a simple conduit for food transport. In performing this delivery function, it confronts a continuous influx of foreign antigens, including food particles with variable microbial content, and encounters many biophysical stimuli triggered by food textures and temperature. To meet these challenges, the oesophagus comprises a robust barrier featuring a thick, multilayered epithelium coated by mucins and mechanically held together by strong adhesion complexes, including desmosomal junctions. Sentinel immune cells, including a notable presence of CD8+ resident memory T cells, mast cells and dendritic cells, are abundant alongside IL-1 family cytokines released and activated under tight homeostatic regulation through a balance of proteases and antiproteases. Pattern recognition receptors, such as Toll-like receptors on epithelial cells, identify foreign antigens and can trigger cytokine release. Disruptions, whether acquired or genetically inherited, in these innate immune functions contribute to disease onset. Here, we present evidence that the oesophagus is an immune organ with extensive sensing properties designed to tolerate and mount defences against antigenic and biophysical challenges. This Perspective article provides a detailed overview of the oesophagus as an immune organ, highlighting its innate immune components and sensing mechanisms that maintain homeostasis and barrier integrity while responding to antigenic and biophysical challenges.

The esophagus has long been regarded as a simple conduit for food transport. A new paradigm now recognizes it as an immune organ actively involved in environmental sensing, antigen tolerance, and neuroimmune signaling to maintain mucosal homeostasis. The stratified squamous epithelium provides a vital physical barrier, supported by transmembrane mucins and regulated by posttranslational modifications, including citrullination mediated by esophagus-enriched peptidyl arginine deiminases. A unique esophageal microbiome, established early in life, shapes epithelial differentiation, gene expression, and immune development and response. Pattern recognition receptors and enriched IL-1 family cytokines, including the alarmins IL-33 and thymic stromal lymphopoietin, orchestrate rapid immune responses to diverse environmental stimuli, such as allergens. The protease-antiprotease balance, controlled by serine and cysteine proteases, notably kallikreins, and the inhibitors SERPINs and SPINKs, regulates cytokine activity and barrier integrity. Neuroimmune circuits link innate immune activation, sensory perception, and barrier regulation. Disruption of these pathways, including genetic variants in barrier components, proteases, and immune regulators, contributes to disease susceptibility and pathogenesis, most notably eosinophilic esophagitis. Thus, the esophagus is now understood to be an environmental sensing immune organ with functions in health and disease.

Eosinophilic esophagitis (EoE) is a chronic allergic inflammatory disease of the esophagus. Single-cell RNA sequencing has shown esophageal mast cell activation in active EoE and remission. We hypothesized that there is esophageal mast cell degranulation in EoE patients with active and remission disease states.

The interplay between genetic and environmental factors during pregnancy can predispose to inflammatory diseases postnatally, including eosinophilic esophagitis (EoE), a chronic allergic disease triggered by food. Herein, we examined the effects of amniotic fluid (AF) on esophageal epithelial differentiation and responsiveness to proallergic stimuli. Multiplex analysis of AF revealed the expression of 66 cytokines, whereas five cytokines including IL-4 and thymic stromal lymphopoietin (TSLP) were not detected. Several proinflammatory cytokines including TNFα and IL-12 were highly expressed in the AF from women who underwent preterm birth, whereas EGF was the highest in term birth samples. Exposure of esophageal epithelial cells to AF resulted in transient phosphorylation of ERK1/2 and the transcription of early response genes, highlighting the direct impact of AF on esophageal epithelial cells. In a three-dimensional spheroid model, AF modified the esophageal epithelial differentiation program and enhanced the transcription of IL-13-target genes, including CCL26 and CAPN14, which encodes for a major genetic susceptibility locus for eosinophilic esophagitis. Notably, CAPN14 exhibited upregulation in spheroids exposed to preterm but not term AF following differentiation. Collectively, our findings call attention to the role of AF as a potential mediator of the intrauterine environment that influences subsequent esophageal disorders.NEW & NOTEWORTHY The interaction between amniotic fluid and the esophageal epithelium during pregnancy modifies esophageal epithelial differentiation and subsequent responsiveness to inflammatory stimuli, including interleukin 13 (IL-13). This interaction may predispose individuals to inflammatory conditions of the esophagus, such as eosinophilic esophagitis (EoE), in later stages of life.

BackgroundEosinophilic esophagitis (EoE) is diagnosed and monitored using esophageal eosinophil levels; however, EoE also exhibits a marked, understudied esophageal mastocytosis.ObjectiveUsing machine learning, we localized and characterized esophageal mast cells to decipher their potential role in disease pathology.MethodsEsophageal biopsy samples (EoE, control) were stained for mast cells by anti-tryptase and imaged using immunofluorescence; high-resolution whole tissue images were digitally assembled. Machine learning software was trained to identify, enumerate, and characterize mast cells, designated Mast Cell-Artificial Intelligence (MC-AI).ResultsMC-AI enumerated cell counts with high accuracy. During active EoE, epithelial mast cells increased and lamina propria (LP) mast cells decreased. In controls and EoE remission patients, papillae had the highest mast cell density and negatively correlated with epithelial mast cell density. Mast cell density in the epithelium and papillae correlated with the degree of epithelial eosinophilic inflammation, basal zone hyperplasia, and LP fibrosis. MC-AI detected greater mast cell degranulation in the epithelium, papillae, and LP in EoE patients compared with control individuals. Mast cells were localized further from the basement membrane in active EoE than EoE remission and control individuals but were closer than eosinophils to the basement membrane in active EoE.ConclusionUsing MC-AI, we identified a distinct population of homeostatic esophageal papillae mast cells; during active EoE, this population decreases, undergoes degranulation, negatively correlates with epithelial mast cell levels, and significantly correlates with distinct histologic features. Overall, MC-AI provides a means to understand the potential involvement of mast cells in EoE and other disorders.

Eosinophilic esophagitis is a chronic food antigen-driven allergic inflammatory disease associated with symptoms involving the nervous system such as refractory pain. Yet, the role of the nervous system in disease pathogenesis has not received much attention. Herein, we demonstrate that allergen exposure evokes pain-like behavior in association with increased nociceptor signaling and transcriptional responses in dorsal root ganglia. NaV1.8+ sensory nerves were found traveling along the length of the esophagus, organized in distinct bundles adjacent to the basal epithelium, with beta III-tubulin+ sensory nerves distributed more distal to the lumen. Targeted deletion of Il4ra in NaV1.8+ neurons impeded allergen-induced increases in nerve innervation density. Furthermore, Il4ra-/-NaV1.8 mice had diminished allergen-induced allergic inflammation in the esophagus including eosinophilia and transcription of pro-inflammatory genes. Translational studies revealed extensive myelinated nerve innervation in the human esophagus, which was increased in patients with eosinophilic esophagitis. Taken together, these data indicate that allergic inflammation is associated with an increase in non-evoked pain, esophageal nerve density, altered sensitivity of sensory neurons, and transcriptional changes in dorsal root ganglia. These finding identify a type 2 neuroimmune circuit that involves the interplay of allergen-induced IL-4 receptor-dependent DRG responses that modify esophageal end-organ inflammatory responses. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND:Eosinophils are elusive cells involved in allergic inflammation. Single-cell RNA-sequencing (scRNA-seq) is an emerging approach to deeply characterize cellular properties, heterogeneity, and functionality. OBJECTIVES:We sought to comprehensively characterize the transcriptome and biological functions of human eosinophils at a site of severe allergic inflammation in the esophagus (ie, eosinophilic esophagitis [EoE]). METHODS:We employed a gravity-based scRNA-seq methodology to sequence blood eosinophils from patients with EoE and control individuals compared to a reanalyzed public scRNA-seq dataset of human esophageal eosinophils of EoE patients. We used flow cytometry, immunostaining, and a stimulation assay to verify mRNA findings. RESULTS:In total, scRNA-seq was obtained from 586 eosinophils (188 from blood [n = 6 individuals] and 398 from esophagus [n = 6 individuals]). The esophageal eosinophils were composed of a population of activated eosinophils (enriched in 659 genes compared with peripheral blood-associated eosinophils) and a small population of eosinophils resembling peripheral blood eosinophils (enriched in 62 genes compared with esophageal eosinophils). Esophageal eosinophils expressed genes involved in sensing and responding to diverse stimuli, most notably IFN-γ, IL-10, histamine and leukotrienes, and succinate. Esophageal eosinophils were most distinguished from other esophageal populations by gene expression of the receptors CCR3, HRH4, SUCNR1, and VSTM1; transcription factors CEBPE, OLIG1, and OLIG2; protease PRSS33; and the hallmark eosinophil gene CLC. A web of bidirectional eosinophil interactions with other esophageal populations was derived. Comparing esophageal eosinophils and mast cells revealed that esophageal eosinophils expressed genes involved in DNAX-activation protein-12 (also known as TYROBP) interactions, IgG receptor-triggered events, immunoregulation, and IL-10 signaling. CONCLUSIONS:In EoE, esophageal eosinophils exist as 2 populations, a minority population resembling blood eosinophils and the other population characterized by high de novo transcription of diverse sensing receptors and inflammatory mediators readying them to potentially intersect with diverse cell types.

Persistent HPV16 infection is a major cause of the global cancer burden. The viral life cycle is dependent on the differentiation program of stratified squamous epithelium, but the landscape of keratinocyte subpopulations which support distinct phases of the viral life cycle has yet to be elucidated. Here, single cell RNA sequencing of HPV16 infected compared to uninfected organoids identifies twelve distinct keratinocyte populations, with a subset mapped to reconstruct their respective 3D geography in stratified squamous epithelium. Instead of conventional terminally differentiated cells, an HPV-reprogrammed keratinocyte subpopulation (HIDDEN cells) forms the surface compartment and requires overexpression of the ELF3/ESE-1 transcription factor. HIDDEN cells are detected throughout stages of human carcinogenesis including primary human cervical intraepithelial neoplasias and HPV positive head and neck cancers, and a possible role in promoting viral carcinogenesis is supported by TCGA analyses. Single cell transcriptome information on HPV-infected versus uninfected epithelium will enable broader studies of the role of individual keratinocyte subpopulations in tumor virus infection and cancer evolution.

Eosinophilic esophagitis (EoE) is a chronic gastrointestinal disorder characterized by food antigen-driven eosinophilic inflammation and hyperproliferation of esophageal mucosa. By utilizing a large-scale, proteomic screen of esophageal biopsies, we aimed to uncover molecular drivers of the disease. Proteomic analysis by liquid chromatography-tandem mass spectrometry identified 402 differentially expressed proteins (DEPs) that correlated with the EoE transcriptome. Immune cell-related proteins were among the most highly upregulated DEPs in EoE compared with controls, whereas proteins linked to epithelial differentiation were primarily downregulated. Notably, in the inflamed esophageal tissue, all 6 subunits of the minichromosome maintenance (MCM) complex, a DNA helicase essential for genomic DNA replication, were significantly upregulated at the gene and protein levels. Furthermore, treating esophageal epithelial cells with a known inhibitor of the MCM complex (ciprofloxacin) blocked esophageal epithelial proliferation. In a murine model of EoE driven by overexpression of IL-13, ciprofloxacin treatment decreased basal zone thickness and reduced dilated intercellular spaces by blocking the transition of epithelial cells through the S-phase of the cell cycle. Collectively, a broad-spectrum proteomic screen has identified the involvement of the MCM complex in EoE and has highlighted MCM inhibitors as potential therapeutic agents for the disease.

BACKGROUND:Eosinophilic gastritis (EoG) associates with type 2 immunity. However, the type 2 cytokine cellular source, gastric T-cell composition, and gastric T-cell relationship (or relationships) with disease pathology remain understudied. OBJECTIVE:We defined gastric T-cell populations and their association with histologic and endoscopic EoG pathology. METHODS:Gastric biopsy samples (n = 6 EoG, n = 7 control) were subjected to histologic, endoscopic, and flow cytometry analyses. In a complementary cohort (n = 83 EoG), IL4, IL5, and IL13 mRNA levels were correlated with EoG pathologic parameters. RESULTS:Gastric biopsy samples contained CD3+ T cells that were mainly CD8+; the CD8/CD4 ratio was comparable in EoG and control biopsy samples (5.7 ± 3.0 and 4.3 ± 0.6, respectively; P = .28). Gastric regulatory T (CD3+CD4+FOXP3+) and TH2 (CD3+CD4+GATA3+) cell levels were increased in EoG versus controls (2-fold, P < .05 and 10-fold, P < .001, respectively) and correlated with gastric eosinophil levels (r = 0.63, P < .05 and r = 0.85, P < .001, respectively), endoscopic pathology (r = 0.56, P < .01; r = 0.84, P < .001, respectively), and histopathology (r = 0.72, P < .01; r = 0.82, P < .01, respectively). Cytokine-positive, most notably IL-4+, TH2 cell levels strongly correlated with histologic and endoscopic scores (r = 0.82, P < .0001 and r = 0.78, P < .0001, respectively). In an independent EoG cohort (n = 83), bulk gastric IL4, IL5, and IL13 mRNA levels correlated with histologic score (r = 0.22, P < .005; r = 0.54, P < .0001; and r = 0.36, P < .0001, respectively) and endoscopic score (r = 0.27, P < .001; r = 0.40, P < .0001; and r = 0.35, P < .0001, respectively). CONCLUSIONS:EoG is a TH2 cell-associated disease featuring increased gastric type 2 cytokine-producing CD3+CD4+GATA3+TH2 cells that strongly correlate with disease pathologies.

Eosinophilic esophagitis (EoE) is a type 2 allergic disease characterized by esophageal inflammation and epithelial cell dysfunction. Acquired loss of the anti-serine protease of kazal type 7 (SPINK7) in the squamous epithelium of the esophagus has a causal role in EoE pathogenesis. Yet there is a limited understanding of the factors that regulate its expression and responsiveness to inflammatory stimuli. Herein, we identified the transcription factor, ovo like transcriptional repressor 1 (OVOL1) as an esophageal selective gene product that regulates SPINK7 promoter activity. Overexpression of OVOL1 increased SPINK7 expression, whereas, its depletion decreased SPINK7 expression, impaired epithelial barrier and increased production of the pro-atopy cytokine thymic stromal lymphopoietin (TSLP). Mechanistically, ligands of AHR induced nuclear translocation of OVOL1 which in turn promoted epithelial cell differentiation, barrier function and SPINK7 expression. Interleukin (IL)-4 and IL-13 abolished AHR ligand-induced OVOL1 nuclear translocation. Stimulation with IL-13 abrogated the nuclear translocation of OVOL1 and promoted enhanced degradation of OVOL1 protein. This effect of IL-13 was dependent on the esophageal specific cysteine protease calpain-14. Translational studies demonstrated loss of OVOL1 protein expression in patients with EoE. In summary, AHR mediates its action via OVOL1-induced SPINK7 transcription, and IL-4 and IL-13 repress this pathway in EoE. As such, activation of the AHR pathway is a potential intervention strategy for reversing EoE. Graphical abstract The influence of the exposome on regulatory networks in EoE pathogenesis. AHR is activated and influenced by diet nutrients, environmental toxicants, microbiome composition, tryptophan metabolites, and drugs. When AHR is activated, it promotes translocation of OVOL1 to the nucleus, which in turn promotes expression of epithelial genes including SPINK7 . SPINK7 expression promotes epithelial differentiation, barrier function, decreased proteolytic activity, and decreased TSLP production. IL-4 and IL-13 inhibit OVOL1 nuclear translocation and therefore, repress SPINK7 expression. IL-13–stimulated CAPN14 expression decreases OVOL1 protein expression and SPINK7 transcription.