An integrative single-cell and spatial transcriptomic analysis of fistulating Crohn's disease

Thesis

Crohn’s disease (CD) often leads to fistula development in approximately 40% of patients, driven by sustained, transmural inflammation of the bowel wall. Despite advanced treatments, one-third of patients experience fistula recurrence, highlighting a significant clinical challenge. The pathogenesis of fistulating CD remains poorly defined and is considered multifactorial, involving complex interactions among immune responses, cellular dynamics, genetic factors and microbial contributions. While processes such as epithelial-to-mesenchymal transition, upregulation of matrix metalloproteinases, pro-inflammatory markers like TNF-alpha and IL-13 and NOD2 polymorphisms have been implicated, comprehensive molecular studies of fistulating CD are lacking, limiting the development of targeted therapies.

This project addresses this gap by investigating the mechanisms underlying diverse CD fistulae presentations using integrative multimodal single cell RNA-sequencing and spatial transcriptomics (ST). Subcellular resolution in situ ST, alongside full-transcriptome ST profiling, was applied to over 60 fistulae and more than 6 million imaged cells, revealing common pathological features across various CD fistula phenotypes. The analysis identified novel fibroblast subpopulations at the leading edge of fistulae, defined by patterning-associated transcription factors and driven by dysregulated morphogen networks in both epithelialised and non-epithelialised fistulae. Additionally, a high-resolution view of the re-epithelialisation process within fistulae and the impaired stromal-epithelial crosstalk that hinders epithelial regeneration, was elucidated.

The findings offer a detailed multimodal atlas of fistulating CD at an unprecedented resolution, serving as a key resource for developing disease models and identifying novel therapeutic targets. This work represents a significant advance in understanding the molecular and cellular mechanisms driving fistula formation in CD, with the potential to inform future strategies aimed at improving patient outcomes.

Authors: McGregor, C

• DOI: 10.5287/ora-vjn5rjao5
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: spatial transcriptomics; single-cell RNA-sequencing; pathogenesis; fistulae; Crohn's disease; fibroblasts
• Subjects: Immunology; Inflammatory bowel diseases; Pathogenesis