Effects of C3aR activated mast cells in eosinophilic esophagitis

Abstract

BackgroundEosinophilic 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. MethodsEsophageal 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. ResultsEsophageal 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. ConclusionTaken together, our findings present evidence for the interplay of mast cells and fibroblasts via C3a in the pathogenesis of EoE.