WOFAPS 2025 8th World Congress of Pediatric Surgery

Dengming Lai, Jingyi Jin, Linghao Cai, Jinfa Tou, Qiang Shu

Children's Hospital Zhejiang University School of Medicine, Hangzhou, China

Background: Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease that predominantly affects premature neonates and is associated with high morbidity and mortality. Macrophages play a critical role in NEC pathogenesis; however, due to their high heterogeneity, the precise mechanisms by which they influence disease progression remain unclear.

Methods: Single-cell RNA sequencing (scRNA-seq) was performed on intestinal tissues from five NEC patients and four controls. Cell clusters were identified based on canonical marker genes, and subsequent analyses included differential gene expression and pathway enrichment. Mass cytometry (CyTOF) was used to characterize immune cell subsets in both intestinal tissues and peripheral blood during NEC. Triggering receptor expressed on myeloid cells 2 (TREM2) expression was assessed using imaging techniques and Western blotting. NEC models were established in both wild-type (WT) and Trem2 knockout mice to evaluate survival, intestinal injury, and intestinal stem cell (ISC) dynamics.

Results: scRNA-seq analyses revealed a significant reduction in a macrophage subpopulation marked by high TREM2 expression. Based on CyTOF analysis, CD45⁺ immune cells were classified into 27 distinct subsets, while macrophages represented the most prominently increased in both intestinal tissues and peripheral blood in the NEC. Moreover, macrophages were devided into 11 subsets (M01–M11). Among them, subset M11 was the predominant macrophage population in peripheral blood, while M10 represented a tissue-specific subset that was significantly increased following NEC onset. In contrast, M02, which was highly abundant in tissue, and M08, which was predominant in peripheral blood, were both markedly reduced after NEC development. Differential gene expression analysis indicated that TREM2 plays a key regulatory role. TREM2 expression was markedly decreased in intestinal tissues from both NEC patients and NEC-induced mice. Trem2 knockout mice exhibited lower survival rates and more severe intestinal injury compared to WT controls. Additionally, a marked reduction in ISCs was observed following NEC, which was significantly more pronounced in Trem2 knockout mice. In vitro experiments demonstrated that TREM2-high macrophages support the proliferation of ISCs.

Conclusions: TREM2-high macrophages are significantly depleted during NEC and play a protective role in maintaining intestinal integrity by promoting intestinal stem cell proliferation.