Multi-layer transcriptomic analyses identify a mucin-associated epithelial program linked to innate inflammatory injury in ulcerative colitis.

Epithelial barrier disruption and mucosal innate immune activation reinforce each other in ulcerative colitis (UC), yet the epithelial glycosylation programs linking mucus homeostasis to inflammatory tissue injury remain incompletely defined. Here, we integrated bulk, single-cell, and spatial transcriptomic analyses with functional validation to identify mucin-associated epithelial determinants relevant to UC. In the discovery cohort GSE107499, 15 dysregulated mucin-type O-glycosylation and mucin-associated genes were identified and were enriched mainly in O-glycan biosynthesis, glycosyltransferase activity, and Golgi-associated secretory functions. An integrative prioritization framework highlighted GALNT12 as the leading candidate. Low GALNT12 expression was associated with enrichment of TNFA signaling via NF-kB, inflammatory response, interferon gamma response, IL6-JAK-STAT3 signaling, and complement, together with higher infiltration scores for monocyte lineage cells, neutrophils, fibroblasts, and T-cell populations. At single-cell resolution, GALNT12 was concentrated in epithelial cells, particularly MUC2-positive epithelial cells. MUC2-positive epithelial cells with detectable GALNT12 expression showed broader inferred incoming and outgoing communication with surrounding epithelial, stromal, and immune populations and were enriched for proteostasis and secretory trafficking programs. Spatial transcriptomic analysis further identified GALNT12-high mucin-associated niches that co-localized with stronger goblet and mucin signatures and lower inflammatory scores. In HT29-19A cells, GALNT12 knockdown exacerbated TNF-α-induced LDH release, reduced OCLN and TJP1 expression, increased IL-8, IL-6, CCL2, IL-1β, and IL-18 production, and enhanced caspase-1 processing detected as cleaved caspase-1 p10 together with increased GSDMD-N formation under TNF-α stress. Collectively, these findings identify a GALNT12-centered mucin-associated epithelial program linked to epithelial barrier resilience and relatively restrained innate inflammatory injury at the barrier-immune interface in UC, while supporting a caspase-1/GSDMD-associated inflammatory injury phenotype under cytokine stress.