Proteomic analysis of chronically inflamed intestine and disease-related strictures in Crohn’s disease patients

Abstract

Crohn’s disease (CD) is a chronic inflammatory bowel disease characterized by recurrent episodes of dysregulated immunity in segments of the gastrointestinal tract, leading to progressive tissue damage. Despite advances in medical therapies to reduce inflammation, many patients experience lifelong suffering and undergo repeated surgeries due to long-term complications, with no cure currently available. One severe CD complication is intestinal strictures, characterized by muscular hypertrophy and fibrosis in chronically inflamed intestine, causing successive narrowing and obstruction. Despite its severity, the mechanism underlying stricture formation remains poorly understood. This thesis advances understanding of stricture-associated mechanisms in CD using unbiased mass spectrometry and targeted spatial proteomics. In the first study, we identified proteome signatures in FACS-sorted immune- and epithelial cells from inflamed mucosal tissue from CD patients compared to controls. Proteins linked to mitochondrial translation and neutrophil degranulation were enriched in immune cells from CD mucosa, while epithelial cells showed prominent changes in glycosylation and secretory pathways. The second study examined CD patients with intestinal strictures and compared the proteomes of laser-microdissected submucosa (SM) and muscularis propria (MP) tissue layers in stricture versus control. Significant proteome shifts were observed in both tissue layers, with more pronounced alterations in the SM. In the SM, a transition from homeostasis to inflammation, fibrosis and muscular expansion characterized the proteome of strictures versus controls. Differential expression (DE) analysis revealed distinct protein clusters associated with each of these pathological states in stricture SM. In the MP layer, increases in immunity and ECM-associated proteins were accompanied by reductions in mitochondrial and key muscle regulatory proteins. Beyond layer-specific alterations, interlayer analysis highlighted concordant changes in subsets of proteins in the SM and MP, suggesting parallel processes occur in these layers in stricture formation. Top increased DE proteins shared between the layers included immune-related proteins and ER chaperones, while top shared decreased DE proteins were lipid transporters. Building on these insights, spatial proteomics was used for cellular profiling of strictured intestinal wall. Distinct spatial distributions and cellular associations of top DE proteins were apparent in strictured tissue, suggesting their potential roles in pathogenesis. In conclusion, this thesis provides detailed protein-level data and cellular profiling of defined anatomic regions of strictured intestine, an area that thus far is largely unexplored. The insights gained through this exploratory approach reveal several protein targets potentially involved in stricture pathogenesis, and opens avenues to explore new treatments for CD-related strictures. Finally, we show that differential protein expression is spatially restricted, emphasizing the importance of compartmentalized analysis of human tissue to optimize drug development.