LILRB3 as an ulcerative colitis-associated molecular biomarker: integrated transcriptomic, single-cell, and experimental validation.

Objective

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by mucosal inflammation and immune dysregulation. Although macrophage-mediated inflammation is associated with UC progression, its molecular basis remains incompletely understood. This study integrated bioinformatic analyses and experimental validation to identify UC-associated molecular biomarkers and explore their roles in immune dysregulation.

Methods

Bulk transcriptomic datasets were integrated for differential expression analysis, weighted gene co-expression network analysis (WGCNA), and multi-algorithm machine learning. The machine-learning model was evaluated in independent validation cohorts, and a nomogram was subsequently constructed and evaluated in an additional colonic-tissue cohort. Immune infiltration, single-cell RNA sequencing, cell-cell communication, pseudotime analysis, and scTenifoldKnk-based virtual knockout were used to investigate hub genes. RT-qPCR, WB, and IHC validated the findings in a DSS-induced mouse model and clinical samples, while siRNA-mediated LILRB3 knockdown was performed in THP-1-derived macrophages.

Results

Differential expression analysis and WGCNA identified 55 candidate genes. Through screening of 127 machine-learning algorithm combinations, DPP10, LILRB3, MTMR11, and SLC6A14 were identified as UC-associated molecular biomarkers. A separately constructed logistic regression nomogram showed an apparent AUC of 0.989. LILRB3 was predominantly expressed in myeloid cells, particularly M1 macrophages, and was associated with inflammatory immune cell infiltration. Cell-cell communication analysis revealed that LILRB3⁺ M1 macrophages exhibited active intercellular communication involving MHC-II-CD4 and other inflammation-related signaling pathways. Experimental validation showed increased PirB expression in the colonic tissues of DSS-induced colitis mice and elevated LILRB3 expression in human UC colonic tissues. Knockdown of LILRB3 in THP-1-derived macrophages significantly reduced TNF-α, IL-6, and IL-1β mRNA expression.

Conclusion

LILRB3 is a candidate UC-associated molecular biomarker in colonic tissue and may be associated with proinflammatory macrophage remodeling and myeloid immune dysregulation, warranting mechanistic investigation.