Background: Celiac disease (CeD) is characterized by gluten-driven immune activation and epithelial injury, yet the spatial molecular events before villous atrophy remain poorly understood. Potential celiac disease (PCeD), defined by celiac-specific autoantibodies in the absence of villous atrophy, provides a unique opportunity to investigate these early disease mechanisms. We applied GeoMx Digital Spatial Profiling (DSP) to characterize compartment-specific epithelial transcriptional changes across disease progression.
Methods: Duodenal biopsies from patients with CeD (n = 10), PCeD (n = 2), and healthy controls (n = 5) were analyzed using GeoMx DSP. Regions of interest were selected from crypt and villus compartments and segmented into epithelial and lamina propria areas. Following quality control and normalization, differential gene expression and pathway enrichment analyses were performed. Key findings were validated by RNAscope in situ hybridization and immunofluorescence.
Results: Principal component analysis demonstrated that disease-associated transcriptional changes were most pronounced in epithelial compartments, which therefore became the primary focus of subsequent analyses. Active CeD was characterized by induction of antigen presentation, interferon signaling, and epithelial stress-response pathways. HLA class I-associated genes, including HLA-E and HLA-F, together with interferon-responsive genes, were already increased in PCeD, whereas robust induction of HLA class II genes (HLA-DRB1, HLA-DQB1) was observed predominantly in overt CeD, indicating stepwise activation of epithelial immune programs during disease progression. RNAscope confirmed marked epithelial IL32 expression in CeD, while immunofluorescence demonstrated epithelial expression of HLA-B and HLA-E consistent with the spatial transcriptomic findings.
Conclusions: Spatial transcriptomics reveals progressive epithelial immune activation during the transition from PCeD to overt CeD. Early activation of HLA class I- and interferon-associated pathways precedes the emergence of epithelial HLA class II programs, providing new insight into the molecular events underlying progression to active disease.