肠道共生菌F.prausnitzii胞外囊泡促进Treg细胞分化抑制IBD肠道炎症的机制研究

Gut microbiota dysbiosis plays important role in the pathogenesis of inflammatory bowel diseases (IBD). Normal gut microbiota can regulate the proliferation and differentiation of intestinal Treg cells to maintain intestinal immune homeostasis. The effect of F. prausnitzii (Fp) in the regulation of intestinal immunity in IBD is unclear. Our previous data showed that Fp was significantly decreased in IBD patients compared to the healthy subjects, and extracellular vesicles (EVs) isolated from Fp culture induced CD4+Foxp3+ Treg cell differentiation and IL-10 production. We hypothesized that the Fp secreted EVs carrying bacterial components promotes CD4+Foxp3+ Treg cell differentiation and proliferation, resulting in IL-10 production and inhibits intestinal inflammation. We will firstly examine the effect of Fp EVs on intestinal inflammation in DSS induced experimental colitis in mice. Secondly, we will co-culture Fp EVs with naive mice CD4+ T cells to analyze the impact of Fp EVs on CD4+Foxp3+ Treg cell differentiation. Finally, we will investigate the mechanisms of the induction of CD4+Foxp3+ Treg cell differentiation by Fp EVs through blocking toll-like receptor 2 (TLR2) signaling in CD4+ T cells in vitro and TLR2 knockout mice in vivo. Our study will provide experimental evidences to reveal the roles of Fp in the pathogenesis of IBD, and suggest the potential application of bacteria EVs-based treatment in the future.

肠道菌群失调是炎症性肠病（IBD）始动和持续的重要因素。正常肠道菌群可调节肠道Treg细胞分化来维持肠道免疫稳态。肠道共生菌F.prausnitzii（Fp）在IBD中的免疫调控作用尚不明确。我们前期研究显示Fp在IBD患者中明显减少，且Fp源胞外囊泡（EVs）可诱导小鼠Treg细胞分化，促进IL-10表达。我们假说Fp源EVs可传递细菌抗原诱导CD4+Foxp3+Treg细胞分化，产生IL-10抑制炎症。本项目将以DSS诱导小鼠结肠炎模型来研究Fp源EVs对肠道炎症的抑制作用；将Fp源EVs与小鼠脾脏和淋巴结CD4+T细胞共培养，研究Fp源EVs对CD4+Foxp3+T细胞分化的作用；通过阻断CD4+T细胞TLR2和TLR2基因敲除小鼠探讨Fp源EVs诱导CD4+Foxp3+T细胞分化的分子机制，为揭示Fp在IBD中的作用机制提供理论基础，为开发基于肠道菌群EVs的治疗方法提供实验依据。

肠道菌群失调是炎症性肠病（IBD）始动和持续的重要因素。正常肠道菌群可调节肠道Treg细胞分化来维持肠道免疫稳态。在DSS诱导结肠炎前，给予C57BL/6小鼠口服Fp-EVs (100 ~ 400 μg) 5天。分析体重、粪便状况、结肠长度和组织病理学。RT-PCR和ELISA检测促炎细胞因子。Western blotting检测结肠组织中的紧密连接蛋白(TJs)。流式细胞术检测固有层淋巴细胞(LPLs)中调节性T细胞(Treg)比例。超离分离得到的Fp-EVs呈典型的双凹盘状，平均直径172 nm。在动物实验中，Fp-EVs治疗可减少dss诱导的小鼠体重减轻、疾病活动指数(DAI)评分、结肠长度缩短、组织学损伤和中性粒细胞浸润。Fp-EVs可降低DSS治疗小鼠促炎细胞因子、白细胞介素-6 (IL-6)、IL-1β和肿瘤坏死因子(TNF)-α的表达。Fp-EVs可上调结肠炎小鼠结肠组织中occludens (ZO)-1、Occludin蛋白的表达，增加Treg细胞的比例。我们的研究结果表明，Fp-EVs通过调节肠黏膜屏障功能和免疫特性来减弱dss诱导的结肠炎。