Mark Rochman

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.

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.

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.

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.

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.

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.

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 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.

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.

Objective: The contribution of vitamin D (VD) deficiency to the pathogenesis of allergic diseases remains elusive. We aimed to define the impact of VD on oesophageal allergic inflammation.Design: We assessed the genomic distribution and function of VD receptor (VDR) and STAT6 using histology, molecular imaging, motif discovery and metagenomic analysis. We examined the role of VD supplementation in oesophageal epithelial cells, in a preclinical model of IL-13-induced oesophageal allergic inflammation and in human subjects with eosinophilic oesophagitis (EoE).Results: VDR response elements were enriched in oesophageal epithelium, suggesting enhanced VDR binding to functional gene enhancer and promoter regions. Metagenomic analysis showed that VD supplementation reversed dysregulation of up to 70% of the transcriptome and epigenetic modifications (H3K27Ac) induced by IL-13 in VD-deficient cells, including genes encoding the transcription factorsHIF1AandSMAD3, endopeptidases (SERPINB3) and epithelial-mesenchymal transition mediators (TGFBR1, TIAM1, SRC, ROBO1, CDH1). Molecular imaging and chromatin immunoprecipitation showed VDR and STAT6 colocalisation within the regulatory regions of the affected genes, suggesting that VDR and STAT6 interactome governs epithelial tissue responses to IL-13 signalling. Indeed, VD supplementation reversed IL-13-induced epithelial hyperproliferation, reduced dilated intercellular spaces and barrier permeability, and improved differentiation marker expression (filaggrin, involucrin). In a preclinical model of IL-13-mediated oesophageal allergic inflammation and in human EoE, VD levels inversely associated with severity of oesophageal eosinophilia and epithelial histopathology.Conclusions: Collectively, these findings identify VD as a natural IL-13 antagonist with capacity to regulate the oesophageal epithelial barrier functions, providing a novel therapeutic entry point for type 2 immunity-related diseases.