Background/Purpose: Esophageal involvement is one of the strongest predictors of early mortality in individuals with systemic sclerosis (SSc). Individuals with SSc and esophageal involvement typically suffer from chronic acid reflux and dysphagia due to loss of esophageal motility. However, the pathogenesis of esophageal dysmotility in SSc is still poorly understood.

Recently, studies have demonstrated that esophageal epithelial cells (EECs) in the mucosa may play a more central role in the pathogenesis of SSc esophageal dysmotility than previously thought. Most studies implicating EECs, however, have been performed in mice or in vitro, and molecular analyses in humans have so far been limited to bulk tissues using microarrays with inadequate controls. In this study, we performed a thorough transcriptomic investigation of the SSc esophageal epithelium in humans using single-cell RNA sequencing (scRNA-seq) to determine whether distinct molecular and cellular changes in EECs contribute to impaired motility of the esophageal wall in SSc.

Methods: We performed scRNA-seq of paired proximal and distal esophageal mucosa biopsies from 10 individuals with SSc and 6 healthy controls (HCs). We also evaluated samples from 4 individuals with gastroesophageal reflux disease (GERD) to distinguish primary SSc effects from secondary reflux effects. We further assessed esophageal motility (normal, weak, absent) in individuals with SSc using functional lumen imaging probe panometry and high-resolution manometry. Cells were encapsulated using the 10X Genomics Chromium platform and sequenced on the Illumina Novaseq 6000. Reads were aligned and quantified using Cell Ranger v6.1.2 and cell-specific gene expression was analyzed using Seurat v4.3.0.

Results: ScRNA-seq generated 12.4 billion reads from 434,582 cells across 40 sequenced samples. Following quality control, we annotated 230,720 EECs according to their differentiation state (basal, suprabasal, superficial). Overall proportions of basal, suprabasal, and replicating EECs were similar across conditions, but SSc and GERD samples had significantly fewer terminally differentiated, superficial cells than HCs. Differential gene expression analyses revealed dysregulated gene sets dependent on condition, biopsy region, and EEC layer. A surge of gene dysregulation in the transition from suprabasal to superficial states was observed in both SSc and GERD. We then modeled EEC differentiation as a continuous metric based on cumulative average expression of layer-specific markers and identified genes with condition-dependent expression trends as a function of EEC differentiation, highlighting genes uniquely dysregulated in SSc, including those correlated with esophageal dysmotility.

Conclusion: Esophageal dysmotility in SSc is associated with cellular and molecular changes in EECs distinct from secondary reflux effects. This work serves as an atlas for the human esophageal epithelium in SSc to direct future efforts to identify actionable targets for treatment.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mapping-cellular-landscape-of-esophageal-epithelium-in-systemic-sclerosis-using-single-cell-transcriptomics/