IL1B-high macrophages promote ferroptosis-like lipid peroxidation injury in Crohn's disease intestinal epithelium.

Crohn's disease (CD)-associated mucosal injury involves immune-cell infiltration, epithelial cell death, and junctional disruption, but how specific myeloid states contribute to epithelial lipid peroxidation remains unclear. We integrated longitudinal single-cell transcriptomics, spatial transcriptomics, macrophage-epithelial co-culture, epithelial RNA-seq, exploratory candidate-gene prioritization, GPX4 functional validation, and a TNBS-induced chronic colitis model to investigate IL1B-high macrophage-associated epithelial injury. In paired pretreatment and post-treatment biopsies, persistent active disease (POST-ACT) showed a higher relative representation of myeloid cells, increased epithelial programmed-cell-death-related transcriptional activity, and stronger predicted myeloid-epithelial communication than remission samples. Myeloid subclustering identified a pro-inflammatory IL1B-high macrophage state relatively enriched in pooled POST-ACT cells, while RCTD-based spatial mapping supported non-random local co-occurrence of inferred IL1B-high macrophage and injury-associated epithelial signals. In vitro, IL1B-high macrophage co-culture increased epithelial cell death, reduced GSH, and increased MDA and lipid ROS in FHC and NCM460 cells; these changes were partially attenuated by Ferrostatin-1. Integration of ferroptosis-related genes, differential expression, LASSO, and random forest prioritized GPX4, PGD, and LPCAT3. GPX4 was consistently downregulated and was selected for functional validation; GPX4 overexpression partially restored ZO-1, E-cadherin, and Occludin expression. Blocking IL-1β/IL1R1 signaling with an anti-IL-1β neutralizing antibody or the IL1R1 antagonist AF12198 attenuated lipid peroxidation, GPX4 downregulation, and loss of junction-associated molecules in the shared co-culture system. In TNBS colitis, Ferrostatin-1 or anti-IL-1β treatment alleviated selected mucosal injury phenotypes, and adding IL-1β blockade to anti-TNF produced incremental improvement over anti-TNF alone. Collectively, these findings support an IL1B-high macrophage-associated epithelial injury axis involving IL-1β-IL1R1 signaling, reduced GPX4-related lipid-peroxide defense, ferroptosis-like lipid peroxidation injury, and junction-associated molecular alterations in CD.