窗口期菌群联合干预调节仔猪肠道菌群和先天免疫系统发育的作用和机制

The immune immaturation and microbial flora dysbiosis are the major reasons for causing early weaning stress in piglets. Promoting the maturation of gut microbial flora and immune may improve piglet growth and effectively control weaning stress. Recent research proves that gut microbial flora synchronously develops with immune system, and early gut colonization with microbiota plays the key role in the development and maturation of innate immune. Moreover, our previous study showed that fecal microbiota transplantation for neonatal piglets significantly improved growth and reduced diarrhea. The hypothesis is that the combined intervention of fecal microbiota transplantation (FMT) and probiotics during the window of opportunity can induce the synchronous development and maturation of gut microbial flora and innate immune, and thereby promotes the growth and reduces weaning stress in piglets. This work carries out with the following parts after the combined intervention of FMT with probiotics during the window of opportunity: 1) investigate the synchronous developmental change of gut microbiota and innate immune system; 2) mining the gut microbiota and functional genes for innate immune development by metagenomic sequencing; 3) screen the specific target genes and metabolic pathways through RNA-seq of innate lymphoid cells in the lamina propria, and thus clarify mechanism of microbiota in regulating development of gut innate immune system. This study may further make a breakthrough on the mechanism of microbiota in regulating gut immune system development, and may also provide a new control strategy for reducing weaning stress in piglets by promoting immune maturation.

免疫不成熟和菌群紊乱是导致仔猪早期断奶应激的重要原因。促进仔猪肠道菌群和免疫成熟，可能促进仔猪生长并有效控制断奶应激。研究证实：肠道菌群与免疫系统协同发育，而菌群在肠道的早期定植对先天免疫发育和成熟起决定性作用；结合我们前期研究发现，对新生仔猪进行母猪粪菌移植，显著促进生长并降低腹泻，提出了假设：在仔猪肠道菌群和免疫发育的窗口期，联合应用粪菌和益生菌干预，可以促进肠道菌群和先天免疫系统协同发育和成熟，促进仔猪生长，缓解断奶应激。拟通过窗口期粪菌联合益生菌干预后，研究仔猪肠道菌群和先天免疫系统的协同变化；通过宏基因组测序挖掘受菌群干预调控并与先天免疫发育密切相关的菌群及功能基因；通过固有层淋巴细胞转录组测序，筛选受菌群干预调控的关键靶基因及代谢通路，阐明肠道菌群调节先天免疫系统发育的分子机制。可能在肠道菌群调控先天免疫系统发育的机制上取得突破，并建立促进肠道免疫成熟缓解仔猪断奶应激的新技术。

仔猪肠道免疫功能不成熟是导致其易发生腹泻等肠道疾病的重要原因。肠道微生物的定值和演替对肠道免疫功能的发育有重要作用。但是，在出生后早期适当的菌群干预能否促进菌群定值并促进肠道免疫发育尚不清楚。本课题证实了利用成熟的母体肠道菌群干预新生仔猪，能显著降低仔猪断奶腹泻率，并显著提高其断奶性能。基于对仔猪肠道菌群从好氧菌到厌氧菌演替的规律，首次提出了利用好氧菌快速创造厌氧环境，同时利用关键厌氧菌加速肠道菌群定值和演替的早期菌群干预方法。筛选了布拉迪酵母菌联合丁酸梭菌的复合干预制剂。揭示了实施早期干预后，仔猪肠道菌群结构更早达到成熟，在7d时已趋向于28d和56日龄，已经具备了较为成熟的代谢碳水化合物、氨基酸等物质的功能。阐明了在早期干预下，菌群结构和功能的改变，可能导致了仔猪在断奶前后肠道先天性免疫和特异性免疫功能的增强，表现为IL-17、IL-22的表达增加，sIgA和疫苗免疫后产生的抗体显著增加，并且肠道的物理屏障功能显著提升。利用靶向代谢组学技术，发现了3日龄和21日龄仔猪粪便的12-HETE的含量最显著差异。通过同源建模、分子动力学模拟和报告基因等方法，揭示了12S-HETE和12R-HETE是Nur77的潜在内源配基，可结合Nur77并激活Nur77的转录调控活性。构建Nur77-/-小鼠，发现Nur77-/-小鼠小肠固有层中 ILC3 的数量和占总淋巴细胞的比例均显著低于WT小鼠，并且对鼠柠檬酸杆菌感染的敏感性增强。而灌注12R-HETE则增加了幼龄小鼠肠道ILC3细胞比例，提高了对鼠柠檬酸杆菌感染的抵抗力。以上结果表明，窗口期菌群联合干预可能通过影响菌群定值和代谢产物的产生，促进了肠道ILC3的发育，提高了仔猪肠道健康水平。