Zheng Ziyi, Lin Lin, Lin Huifang, Zhou Jie, Wang Zhe, Wang Yang, Chen Jianxin, Lai Caimin, Li Renfu, Shen Zhiyong, Zhong Ming, Xie Cheng, Chen Yinjian, Zhang Xuechao, Guo Zhongjie, Dong Rui, He Shiwei, Chen Feng
Department of Pediatric Surgery, Fujian Medical University Union Hospital, Fuzhou, China.
Institute of Population Medicine, School of Public Health, Fujian Medical University, University Town, Fuzhou, China.
Front Immunol. 2025 May 9;16:1559966. doi: 10.3389/fimmu.2025.1559966. eCollection 2025.
Hirschsprung-associated enterocolitis (HAEC) is one of the most severe complications in patients with Hirschsprung's disease (HSCR). Previous research has indicated that acetylcholine (ACH) plays an anti-inflammatory role during inflammation by acting on the α7 nicotinic acetylcholine receptor(α7nAchR) to promote the secretion of anti-inflammatory factors. However, the specific role of ACH in HAEC remains unclear. This experiment aims to explore the sources of ACH in HSCR and its anti-inflammatory mechanisms, thereby identifying new directions for the prevention and treatment of HAEC.
We analyzed single-cell transcriptome data from HSCR to identify cells that secrete ACH and observed their distribution using immunofluorescence. In mice, F4/80, iNOS, ARG-1 and CD206 were used to identify and locate M1 and M2 macrophages in different intestinal segments. Western blot, reverse transcription-quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay were used to test the levels of IκBα, tumor necrosis factor-α, interleukin-10, and the macrophage activation pathway proteins JAK2 and STAT3 in different intestinal segments of mice. Organoid and cell culture techniques were used to verify the anti-inflammatory mechanism of ACH models.
scRNA-seq analysis revealed that tuft cells expressed the CHAT protein. In HSCR, aganglionic segments exhibited heightened cholinergic activity compared with dilated ganglionic segments. In HAEC, inflammation was mainly concentrated in the dilated ganglionic segment and was associated with an increase in M1 macrophages, whereas the aganglionic segment showed less inflammation and was associated with an increase in M2 macrophages. Furthermore, experiments showed that intestinal organoids containing tuft cells promoted an increase in M2 macrophage markers, and ACH promoted M2 macrophage polarization.
Differences in inflammation among various intestinal segments in HAEC may be linked to ACH secreted by tuft cells. Drugs targeting tuft cells have the potential to become important components of HAEC treatment in the future.
先天性巨结肠相关小肠结肠炎（HAEC）是先天性巨结肠（HSCR）患者最严重的并发症之一。先前的研究表明，乙酰胆碱（ACH）在炎症过程中通过作用于α7烟碱型乙酰胆碱受体（α7nAchR）发挥抗炎作用，促进抗炎因子的分泌。然而，ACH在HAEC中的具体作用仍不清楚。本实验旨在探索HSCR中ACH的来源及其抗炎机制，从而为HAEC的防治寻找新方向。
我们分析了HSCR的单细胞转录组数据，以鉴定分泌ACH的细胞，并使用免疫荧光观察其分布。在小鼠中，使用F4/80、诱导型一氧化氮合酶（iNOS）、精氨酸酶-1（ARG-1）和CD206来鉴定和定位不同肠段中的M1和M2巨噬细胞。采用蛋白质免疫印迹法、逆转录定量聚合酶链反应和酶联免疫吸附测定法检测小鼠不同肠段中IκBα、肿瘤坏死因子-α、白细胞介素-10以及巨噬细胞活化途径蛋白JAK2和信号转导和转录激活因子3（STAT3）的水平。使用类器官和细胞培养技术验证ACH模型的抗炎机制。
单细胞RNA测序（scRNA-seq）分析显示，簇状细胞表达胆碱乙酰转移酶（CHAT）蛋白。在HSCR中，无神经节段与扩张的神经节段相比，胆碱能活性增强。在HAEC中，炎症主要集中在扩张的神经节段，且与M1巨噬细胞增多有关，而无神经节段炎症较少，且与M2巨噬细胞增多有关。此外，实验表明，含有簇状细胞的肠类器官促进了M2巨噬细胞标志物的增加，且ACH促进了M2巨噬细胞极化。
HAEC不同肠段的炎症差异可能与簇状细胞分泌的ACH有关。靶向簇状细胞的药物有可能成为未来HAEC治疗的重要组成部分。
