巨噬细胞与固有淋巴细胞相互调控在NEC发病中的作用及机制研究

The intestinal mucosal injury and destruction of the hemostasis of intestinal barrier, which are also considered as the key targets in the clinical treatment for patients with enterocolitis (NEC), are the initiators in the development of NEC. The dysfunction of innate lymphoid cells is shown to be important in induction of the immune responses to cause intestinal injury in patients with NEC. However, the mechanisms remain unknown. Our previous studies and preliminary data showed that polarization of macrophages to M1 promoted inflammatory responses in NEC. Meanwhile, we have also shown that the expression of RORγt, which was considered to be the kernel transcription factor of the type 3 innate lymphoid cells (ILC3), and IL-17 were both increased. Meanwhile, studies showed that, in different microbial environments, the interplays between Mφ and ILC3 could be either immune tolerance or immune injury.Therefore, we speculate that the initiation of Mφ polarization to the pro-inflammatory phenotype leads to transformation of ILC3 to pro-inflammatory phenotypes, followed by cytokine mediated activation and phenotype polarization of Mφ, which finally causes a vicious cycle in the inflammatory responses of NEC. Based on our hypotheses, this project aims to utilize the murine NEC model and in vitro co-culture models to investigate the interplay of Mφ and ILC3 mediated by cytokines, to explore the influence of activation/phenotype polarization of Mφ and subtype transformation of ILC3, to test the expression of associated anti- or pro-inflammatory cytokines, and finally to reveal mechanisms of the vicious cycle in promoting the development of NEC. This study may shed light on the roles of the interplay between macrophages and ILC3 in the development of NEC, provide the new theoretical evidence in the mechanisms of the pathological process of NEC, and predict novel therapeutic targets against treatment of NEC.

肠粘膜损伤及屏障稳态破坏是坏死性小肠结肠炎（NEC）发病的启动因素及治疗难点，而肠道固有免疫细胞功能异常介导的炎症反应是NEC肠道损伤的关键因素，但机制欠明确。我们研究证实新生小鼠NEC病变肠段巨噬细胞（Mφ）向M1型极化，Ⅲ型固有淋巴细胞（ILC3）核心转录因子RORγt和促炎因子IL-17显著增加。同时发现在不同肠道微环境中， Mφ与ILC3相互作用可表现为免疫耐受或损伤。由此推测NEC发生时Mφ向促炎型M1极化，并调控ILC3向促炎表型转化；在细胞因子介导下，ILC3正反馈进一步激活Mφ向M1型极化，导致炎症发展的恶性循环。基于此，本项目拟应用NEC动物及细胞共培养模型，观察Mφ与ILC3活化方向、关键调控细胞因子及对肠上皮的损伤作用机制，探讨细胞因子介导的Mφ与ILC3在促进NEC炎症损伤的双向调控机制，为NEC发病机制的研究提供新的理论依据，为临床诊疗提供新的作用靶点。

肠粘膜损伤及屏障稳态破坏是坏死性小肠结肠炎（NEC）发病的启动因素及治疗难点。研究证实，肠道固有免疫细胞功能异常介导的炎症反应是NEC肠道损伤的关键因素，在不同的肠道微环境中，巨噬细胞（Mφ）与三型固有淋巴细胞（ILC3）相互作用可表现为免疫耐受或损伤，但其与NEC发病的关系尚不明确。我们通过收集临床新生儿NEC组织样本并建立小鼠NEC模型，运用免疫组织化学、Western blotting、rt-PCR、ELISA、TUNEL染色、流式细胞学等多种分子生物学实验方法，在观察了病变肠段的病理损伤、功能变化、细胞凋亡等情况的基础上，检测了Mφ和ILCs的数量、分布、活化方向、分泌细胞因子功能和两者间的联系，并初步探讨了可能的作用机制通路，证实了NEC发生时出现了细胞因子介导的肠道Mφ与ILC3相互调控向促炎表型的转化，其可能与TLR4/NF-κB和JAK/STAT3信号通路的激活有关。本项目研究结果进一步深化了固有免疫介导的炎症反应在NEC发病过程中的重要作用，为NEC发病机制的深入探讨提供了新的理论依据。