Role of NOD2 Variants in Eosinophilic Esophagitis

PROJECT SUMMARY/ABSTRACT Eosinophilic esophagitis (EoE) is a chronic allergic inflammatory esophageal disorder, exhibiting clinical symptoms (e.g., vomiting, pain, dysphagia, food impaction) and histologic features of esophageal eosinophilia, epithelial hyperplasia, and dilated intercellular spaces, which are indicative of impaired barrier function. EoE is highly heritable, yet most genetic research has focused on common variants identified by genome-wide association studies (GWAS), particularly implicating genes like calpain 14 (CAPN14) and thymic stromal lymphopoietin (TSLP), both expressed in esophageal epithelial cells. Our research is a significant extension of these studies, utilizing whole-exome sequencing (WES) on 1,464 patients with EoE from unrelated families, and revealed a notable enrichment of rare genetic variants in the nucleotide-binding oligomerization domaincontaining 2 (NOD2) gene. NOD2 , a key regulator of inflammatory responses, is expressed in both eosinophils and esophageal epithelial cells, suggesting its pivotal role in EoE pathogenesis We central hypothesize that gain-of-function NOD2 variants contribute to esophageal epithelial dysfunction and eosinophil activation, thereby exacerbating inflammatory responses in the type 2 immune environment. Our study comprises three specific aims outlining an integrated set of multidisciplinary studies with the necessary statistical and experimental support to evaluate the impact of the effects of NOD2 variants in EoE. Aim 1 investigates how NOD2 variants impair epithelial barrier integrity, focusing on the mechanistic pathways, including NFKB signaling and reactive oxygen species (ROS) production, that drive dysfunction in esophageal epithelial cells. We will use human induced pluripotent stem cell [iPSC]-derived esophageal organoids with CRISPR/Cas9 genome editing to model the effects of NOD2 variants in epithelial cells. Aim 2 explores the impact of NOD2 variants on eosinophil activation and their interactions with epithelial cells. We hypothesize that these variants enhance eosinophil activation and survival, further amplifying inflammation through altered intercellular signaling. By analyzing the gene expression profiles of cocultured eosinophils and epithelial cells, we aim to clarify how NOD2 variants contribute to EoE pathology via their influence on both cell types. Aim 3 examines the combinatory effects of NOD2 variants with other EoE-associated genetic variants, both common and rare. This aim will help establish whether specific combinations of genetic variants significantly increase the risk for EoE, providing insights into genetic susceptibility and pathogenesis. Our multidisciplinary approach combines genetic, molecular, and functional assays, utilizing advanced disease models, including iPSC-derived gastrointestinal organoids with genome editing of iPSCs by CRISPR/Cas9. This integrative design will provide deeper insights into the genetic mechanisms driving EoE. This research addresses a critical unmet medical need, potentially leading to more personalized and effective treatments for EoE and related gastrointestinal conditions.