干酪乳杆菌环鸟苷腺苷酸通过STING-IFN-λ1信号通路调节树突状细胞分化和成熟的作用机制

Lactobacillus has an important regulatory effect on intestinal mucosal immunity in piglet. It is essential to study the cellular molecular mechanism of intestinal and mucosal immunity with the metabolites of lactic acid bacteria for nutrient absorption and the application of feed supplement. The scientific questions are proposed: the second messenger of cyclic AMP-GMP (c-GAMP) synthesized by Lactobacillus casei was isolated, which activate the STING-IFN-λ1 signal in IPEC, and the expressed IFN-λ1 is secreted into the lamina propria of the intestinal mucosa. The IFN-λ1 regulates the differentiation and maturation of dendritic cells (DC). The CRISPR/Cas9 technology was used to obtain gene knockout cell lines of STING and IFN-λ1, which was used to determine bacterial c-GAMP activating the STING-IFN-λ1 signaling pathway with the Western Blot and laser confocal technology, and elucidate the molecular mechanism of STING-IFN-λ1 signal in IPEC. The secreted IFN-λ1 from IPEC stimulated the DC differentiation and maturation with Transwell model and flow cytometry. The experiments of piglet clarify the regulatory mechanisms of c-GAMP regulating DC differentiation and maturation through the gut. This study aims to explore the mechanism of immunoregulation with Lactobacillus in the cellular level, and provide a theoretical basis for the development and application of lactic acid bacteria in animal husbandry.

乳酸杆菌对仔猪肠黏膜免疫系统具有重要的调节作用，探明乳酸菌代谢产物调节肠黏膜免疫的细胞分子机理，对营养素吸收和饲料添加的合理应用至关重要。拟提出如下科学问题：重点确定干酪乳杆菌的第二信使环鸟苷腺苷酸（c-GAMP）对IPEC的干扰素激活蛋白（STING)-IFN-λ1信号通路的作用机制，以及IFN-λ1分泌到肠黏膜固有层调节树突状细胞（DC）分化和成熟的分子机制。研究利用CRISPR/Cas9技术获得基因敲除STING 和IFN-λ1细胞系，WesternBlot和激光共聚焦阐明c-GAMP激活IPEC的STING-IFN-λ1信号通路作用机制。细胞小室模型和流式细胞术明确分泌到IPEC外的IFN-λ1对DC分化和成熟的细胞分子机制。仔猪实验揭示c-GAMP通过肠道对DC分化和成熟的调控机制。本研究旨在细胞分子水平上阐释乳酸杆菌的黏膜免疫调控机理，为其在畜牧生产中的开发和应用提供理论依据。

乳酸杆菌对仔猪肠黏膜免疫系统具有重要的调节作用，研究主要探寻乳酸菌代谢产物调节肠黏膜免疫的细胞分子机理，对营养素吸收和饲料添加的合理应用至关重要。本课题从干酪乳杆菌的环鸟苷腺苷酸（c-GAMP）对IPEC的干扰素激活蛋白（STING)-IFN-λ1信号通路作用，以及乳酸杆菌基因组表达IFN-λ1分泌到肠黏膜固有层调节树突状细胞（DC）分化和成熟的分子机制：一是从细胞水平上证实益生菌代谢产生的c-GAMP激活肠上皮细胞的STING-IFN-λ1细胞通路，明确STING和IFN-λ1对上皮细胞生长发育的“双刃剑”作用，为合理应用该信号通路在肠道感染免疫中探寻到靶点。第二就是建立乳酸杆菌基因编辑表达平台，表达的IFN-λ1激活肠黏膜免疫细胞的增殖与发育，确定对吞噬细胞中的树突状细胞具有转分化作用。研究为体外应用IFN-λ1生物分子找到最佳的口服递送途径和最佳递送剂量。第三：乳酸杆菌不能简单的定义成微生物与免疫细胞之间的交互作用，研究首次在国际上发现乳酸杆菌逃避巨噬细胞的吞噬作用。并从小鼠体内实验发现乳酸杆菌在小鼠肝脏的定殖作用，在肝脏中激活枯否细胞的免疫活性缓解DSS诱导的结肠炎。并在体外单核巨噬细胞上证实，乳酸杆菌可以结合在巨噬细胞表面增强巨噬细胞活性，但并不能增强巨噬细胞增殖分化。研究为乳酸菌未来在机体健康基础理论研究和应用提出新的视角。