Modulation of NF-kB Signaling by Immunoprobiotics

描述(申请人提供)：这项建议的总体假设是，甘油/甘油磷脂代谢是确定益生菌抑制哺乳动物细胞中核因子？B和MAP激酶信号的相对能力的焦点。最终，关键信号通路的抑制会导致促炎细胞因子的产生下调，并选择性地耗尽激活的免疫细胞。定向和随机突变策略将被用来产生益生菌的插入突变体，这些突变体获得或失去抑制核因子？B激活或MAP激酶信号的能力。为了模拟肠道细菌生态系统，将使用具有特定肠道细菌组合的生物反应器。髓系细胞和上皮细胞将在共培养模型中结合，以提供简化的肠道粘膜模型。乳酸菌产生的主要候选免疫调节因子包括可能下调促炎反应的甘油磷脂衍生物。最后，将选定的益生菌突变体引入到已定义的含有微生物区系的小鼠模型中，以研究已定义的微生物组在体内对核因子？B激活和MAP激酶免疫信号通路的影响。这些研究将在肠道微生物区系具有良好特征的波动的背景下进行，以便可以使用一个小鼠模型并行地研究微生物和宿主对免疫调节的贡献。1.构建免疫益生菌Lactobacillusreuri的插入突变体，筛选调控影响核因子？B和MAPK信号转导途径的关键细菌基因。2.在特定微生物区系的环境中，研究益生菌/突变体对模拟肠粘膜中核因子-βB和MAP激酶信号的调节。3.将野生型和等基因益生菌突变体引入IL-10基因缺陷小鼠体内，研究益生菌对体内核因子-β和MAP激酶信号转导途径的影响。 公共卫生评论：研究人员试图了解有益细菌如何调节肠道炎症。该项目包括对肠道微生物群落的模拟，以便在类似于复杂肠道环境的条件下进行调查。最后，将使用小鼠肠道炎症模型来探索有益细菌的哪些基因对调节哺乳动物的肠道免疫反应是重要的。

DESCRIPTION (provided by applicant): The overall hypothesis of this proposal is that glycerol/glycerophospholipid metabolism is a focal point for determining the relative capacities of probiotics to suppress NF-?B and MAP kinase signaling in mammalian cells. Ultimately, suppression of key signaling pathways results in down-regulation of pro-inflammatory cytokine production, and selective depletion of activated immune cells. Targeted and random mutagenesis strategies will be used to generate insertion mutants of probiotics that gain or lose the ability to suppress NF-?B activation or MAP kinase signaling. Bioreactors with defined combinations of intestinal bacteria will be used in order to simulate gut bacterial ecosystems. Myeloid and epithelial cells will be combined in co-culture models in order to provide simplified models of the gut mucosa. Primary candidate immunoregulatory factors produced by lactobacilli include glycerophospholipid derivatives that may down-regulate pro-inflammatory responses. Finally, selected probiotic mutants will be introduced into a defined microbiota-containing mouse model in order to study effects of a defined microbiome on NF-?B activation and MAP kinase immune signaling pathways in vivo. These studies will occur within the context of well-characterized fluctuations of the intestinal microbiota so that microbial and host contributions to immunoregulation can be investigated in parallel using one mouse model. 1. Generate insertional mutants of immunoprobiotic Lactobacillus reuteri and identify key bacterial genes that regulate factors affecting NF-?B and MAP kinase signaling pathways. 2. Investigate regulation of NF-?B and MAP kinase signaling in a simulated gut mucosa by probiotics/mutants in the milieu of a defined microbiota. 3. Introduce wild type and isogenic probiotic mutants into IL-10-deficient mice with a defined microbiome in order to study probiotic effects on NF-?B and MAP kinase signaling pathways in vivo. PUBLIC HEALTH REVELANCE: The investigator seeks to understand how beneficial bacteria may regulate inflammation in the intestine. This project includes a simulation of the intestinal microbial community so that investigations take place in the context of conditions similar to the complex intestinal environment. Finally, a mouse model of intestinal inflammation will be used to explore which genes of beneficial bacteria are important for regulating intestinal immune responses in mammals.