Contribution of rigid matrix to allergic eosinophilic esophagitis pathogenesis

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Project Abstract Eosinophilic esophagitis (EoE) is a chronic antigen driven type 2 (T2) inflammatory disease affecting up to 1 in 1000 people. EoE requires repeated endoscopy, costing 1.4 billion dollars annually. Chronic untreated or therapy unresponsive EoE leads to progressive esophageal dysfunction due to tissue remodeling and fibrosis. Remodeling causes esophageal rigidity, strictures, clinical dysphagia, food impactions, and failure to thrive. Up to 50% of patients can be non-responsive to standard therapy. Our knowledge gap in treating or reversing fibrosis leaves a dearth of therapies for patients with the greatest need. This situation creates a pressing requirement to understand the mechanisms and cells driving tissue remodeling. Over the last 2 cycles of this R01, we have demonstrated EoE remodeling mechanisms including the effects of TGFβ1 and rigid matrix on esophageal structural cells. We have delineated the EoE inflammatory and remodeling natural history in children. We demonstrated that the EoE fibroblast-derived extracellular matrix (ECM) proteome is unique and sufficient to create a pro-fibrotic myofibroblast phenotype in normal esophageal fibroblasts. These data implicate the EoE fibroblast, itself, as distinct from normal. Single cell RNA sequencing (scRNA-seq) analysis of 3 independent datasets consistently demonstrate 3 transcriptional fibroblast phenotypes that we term secretory (s-), myo-, and inflammatory (i-) fibroblasts. Our proteomic, RNA sequencing, and functional studies demonstrate that the EoE fibroblast has increased type I interferon (IFN-I) response, is more motile, expresses pro-inflammatory CD14, and forms rigid cells. scRNA-seq shows that i-fibroblasts carry an IFN-I response gene pattern. S-fibroblasts are predicted to be collagen producing and pro-inflammatory and be the source for i- and myo-fibroblasts. CD73 is a 5’-nucleotidase that is decreased on EoE s-fibroblasts. CD73 generates anti-inflammatory adenosine and protects from tissue calcification. We propose the central hypothesis that combined decreases in CD73 and increases in CD14 create pathogenic EoE fibroblasts that are poised for rigidity, inflammation, and tissue infiltration. In Aim 1, we will decipher the most pathogenic functions in EoE fibroblasts. We will experimentally investigate the generation of i- and m-fibroblasts from early s-fibroblasts. Using our >15 year longitudinal EoE patient cohort and objective definitions of severity, we will understand which fibroblast phenotypes are retained and lost in severe and mild longitudinal EoE. In Aim 2, we increase and decrease CD73 and CD14 expression and function using transgenes, activating ligands, and a blocking antibody in therapeutic clinical trials. We will understand if loss of fibroblast CD73 aligns with severe and non-remission EoE and if retention of fibroblasts with CD14 are detrimental to EoE course. We uniquely involve a multidisciplinary team, cutting edge technology, and novel computational platforms. For the first time, we will begin to fill our knowledge gap in EoE fibroblast dysfunction which is imperative for controlling esophageal remodeling in children and adults.