Integrative Machine Learning and Single-Cell RNA Sequencing Reveals Timp1+ Stromal Cells as Key Drivers in Ulcerative Colitis.

Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease (IBD). The role of stromal cells in UC pathogenesis is increasingly being recognized, yet their functional subsets and underlying molecular mechanisms remain poorly defined. To integrate multiple transcriptomic datasets, combined weighted gene co-expression network analysis (WGCNA) with machine learning algorithms for the identification of robust core biomarkers of UC and to resolve the cell-subset-specific expression and pathogenic mechanisms of candidate genes using single-cell RNA sequencing (scRNA-seq). Three GEO datasets (GSE38713, GSE3365, and GSE24287) were merged after batch correction. WGCNA, together with five machine learning algorithms, LASSO, random forest, Boruta, gradient boosting machine, and XGBoost, were applied to screen for core genes. External validation was performed using two independent datasets, GSE92415 and GSE179285, and a DSS- of DSS-induced chronic colitis. The cell- subset-specific expression and mechanisms of candidate genes were dissected by scRNA- sequencing. Three core genes, TIMP1, S100A12, and ALPL, were identified. External validation showed that TIMP1 and S100A12 were consistently upregulated in both independent datasets, whereas ALPL was significantly upregulated in GSE92415, but not in GSE179285. TIMP1 was selected for further investigation based on its differential significance. In the DSS-induced chronic colitis mouse model, the area of Timp1-positive colon tissue increased by 2.3-fold compared to in the controls. scRNA-seq revealed that Timp1 was specifically enriched in a stromal cell subset of the mouse colon. Pseudotime trajectory analysis indicated that Timp1⁺ stromal cells predominantly localized to late differentiation stages, with their expression dynamics undergoing a sequential transition from matrix remodeling and angiogenesis to immune regulation. Cell-cell communication analysis further identified the enrichment of the Ppia-Bsg signaling axis between Timp1⁺ stromal cells and epithelial cells. Timp1 was specifically enriched in a colonic stromal cell subset in UC mice, and this subset exhibited a shifted differentiation trajectory and enhanced communication with epithelial cells. These findings provide candidate targets for the molecular diagnosis of UC and highlight the translational potential of targeting Timp1⁺ stromal cells or their downstream signaling pathways for stromal-directed therapeutic intervention.