Paired transcriptomics reveals similarities between cytokine-stimulated organoids and ulcerative colitis epithelial responses

Background and aims: Anti-TNF therapy is central for treating moderate-to-severe ulcerative colitis (UC), yet ~40% of patients are primary non-responders. The molecular basis of non-response in colonic mucosa remains unclear, particularly in a spatial context. We aimed to identify compartment-specific molecular signatures in colonic epithelium and lamina propria distinguishing non-responders from responders with similar baseline disease activity. Methods: UC patients with clear endoscopic response (n=6) or non-response (n=6) after 6 months of anti-TNF therapy were included. Baseline serum cytokines were profiled by multiplex. Pre-treatment inflamed and uninflamed colonic biopsies were laser microdissected into epithelial and lamina propria compartments for RNA sequencing. We explored differentially expressed genes, enrichment and weighted gene co-expression networks. Cellular deconvolution and pseudobulk validation were performed using an independent single-cell atlas, with confirmation by immunohistochemistry. Results: Baseline cytokines showed no significant difference, though responders had higher levels and non-responders greater variability. Inflammation induced more epithelial changes, dominated by interferon signalling and antigen presentation, versus cell cycle and adaptive immune responses in lamina propria. The main differences between responders and non-responders were observed in uninflamed lamina propria. Non-responders more resembled inflamed tissue, showing elevated cell cycle genes and a humoral activity–enriched co-expression network with systematically higher expression of immunoglobulin and B-cell–related genes. The differences disappeared with inflammation. Cellular deconvolution identified expression predominantly in IgA plasma cells, as confirmed through tissue staining and gene expression. Independent pseudobulk analyses validated the anti-TNF non-response-associated pattern. Conclusions: UC uninflamed lamina propria can harbor signatures predictive of anti-TNF non-response, involving cell cycle and humoral immune activation suggesting a TNF-independent immune architecture.