Intestinal Lactobacillus and Mucosal Immunomodulation

描述(由申请人提供)：本提案的中心目标是了解益生菌的分子机制，为炎症性肠病的新治疗和预防策略提供依据。这项建议包括全面的遗传和多肽方法，以确定益生菌抗炎功能的分子基础。我们的实验室已经获得了益生菌的新机制的证据，包括抑制巨噬细胞产生促炎因子TNFa。肠道乳杆菌已被分离并检测肿瘤坏死因子-α(TNF-a)抑制活性。总体假设是，益生菌肠道乳杆菌菌株分泌多肽，抑制巨噬细胞中的肿瘤坏死因子-a信号，抑制肠道炎症。将利用Toll样受体激动剂激活的巨噬细胞，在体外研究调节益生菌活性(包括肽分泌)的候选乳杆菌基因的功能。将野生型乳酸菌和体外缺乏抗炎作用的敲除乳酸菌接种于IL-10基因缺陷小鼠，以探讨体内益生菌的作用机制。识别巨噬细胞中编码抗炎功能的益生菌基因和益生菌反应途径可能为克隆氏病的益生菌作用的潜在人类靶点提供有价值的见解。1.通过基因敲除突变确定肠道乳杆菌中的益生菌抗炎基因。2.通过比较乳杆菌分泌组的多肽性，鉴定抗炎的谷氨酸多肽。3.比较IL-10缺陷小鼠肠道黏膜炎症反应，以敲除或野生型同基因益生菌乳杆菌定植。这些研究将有助于识别可用于治疗炎症性肠道疾病的有益细菌，如克罗恩病和溃疡性结肠炎。我们试图了解有益肠道细菌的作用机制，以便能够自然识别新的菌株或将其用于人类医学。

DESCRIPTION (provided by applicant): The central goal of this proposal is to understand molecular mechanisms of probiosis for new treatment and prevention strategies in inflammatory bowel disease. This proposal includes comprehensive genetic and peptidomic approaches in order to determine the molecular basis of anti-inflammatory functions by probiotics. Our laboratory has obtained evidence for novel mechanisms of probiosis including inhibition of pro-inflammatory TNFa production by macrophages. Intestinal lactobacilli have been isolated and tested for tumor necrosis factor-alpha (TNF-a)-inhibitory activity. The overall hypothesis is that probiotic intestinal Lactobacillus strains secrete peptides that suppress TNF-a signaling in macrophages and inhibit intestinal inflammation. The functions of candidate Lactobacillus genes that regulate probiotic activities including peptide secretion will be studied in vitro using macrophages activated by Toll-like receptor agonists. Wild type lactobacilli and knockout Lactobacillus strains lacking anti-inflammatory effects in vitro will be administered to IL-10-deficient mice in order to explore mechanisms of probiotic action in vivo. Identification of probiotic genes encoding anti-inflammatory functions and probiotic response pathways in macrophages may provide valuable insights into potential human targets for probiotic action in Crohn's disease. 1. Specify probiotic anti-inflammatory genes in intestinal Lactobacillus strains by knockout mutagenesis. 2. Identify anti-inflammatory glutamate-containing peptides by comparative peptidomics of the Lactobacillus secretome. 3. Compare mucosal inflammatory responses in the intestines of IL-10-deficient mice colonized with knockout or wild type isogenic strains of probiotic Lactobacillus. These studies will facilitate the identification of beneficial bacteria that can be used to treat inflammatory bowel disease such as Crohn's disease and ulcerative colitis. We seek to understand the mechanisms of action of beneficial intestinal bacteria so that new strains can be identified naturally or engineered for human medicine.