Bacterial Stress Responses to Intestinal Inflammation

描述（由申请人提供）：克罗恩病和溃疡性结肠炎，统称为炎症性肠病（IBD），部分是由遗传易感宿主对细菌产物的过度侵袭性T细胞介导的免疫反应引起的。大肠杆菌（E. coli）分离物与克罗恩病和细菌成分（包括应激反应蛋白）有关，它们通过刺激先天和适应性宿主免疫系统来促进肠道炎症。虽然对细菌产物的免疫反应已被广泛研究，但对宿主炎症对细菌特性的后续影响知之甚少。与共生的大肠杆菌小鼠菌株（NC101）选择性定植可导致IL-10缺陷（IL-10-/-）小鼠结肠炎，而野生型（wt）小鼠则不会。肠道炎症触发NC101上调细菌应激反应基因ibpB。IbpB是一种小的热休克蛋白，帮助细菌耐受热应激和高水平的活性氧（ROS），如在发炎的肠道和巨噬细胞吞噬溶酶体中发现的。假设：宿主的炎症环境上调了细菌的ibpB，这反过来又使细菌适应环境，并通过增加它们的生存和毒力来延续炎症。具体目的：1)确定宿主炎症环境刺激大肠杆菌上调体外ibpB的机制。我们将利用ibpB- egfp报告菌和ROS和细胞因子产生抑制剂，研究细胞内和分泌因子在上调大肠杆菌ibpB中的作用。2)体外确定大肠杆菌ibpB表达对宿主炎症通路的影响。我们将确定大肠杆菌IbpB抑制巨噬细胞摄取和促进细胞内存活的机制，在庆大霉素保护实验中使用基因改变的NC101和吞噬抑制剂以及ROS生成抑制剂。3)研究大肠杆菌IbpB在大肠杆菌单相关IL-10-/-小鼠慢性实验性结肠炎发生和延续中的生物学相关性。我们将测量与NC101或ibpb缺陷NC101单相关的wt和IL-10-/-小鼠的组织学炎症、细菌分布、先天和适应性免疫反应。职业发展：长期目标是领导学术机构的研究团队：1)为宿主-微生物相互作用领域贡献新知识；2)开发新的IBD诊断策略和治疗方法。短期目标是：1)学习新技术以完成正在进行的和未来的研究；2)定期与优秀的指导团队进行互动；3)参加微生物遗传学和发病机理的教学课程和研究研讨会；4)在细菌应激反应领域发表论文并提交独立资助申请，使自己与导师区别开。环境：国家啮齿动物资源中心、胃肠道生物学和疾病中心、当地粘膜免疫学和细菌学专家、大学启动资金和保护研究时间的机构承诺将促进这些目标的实现。

DESCRIPTION (provided by applicant): Crohn's disease and ulcerative colitis, collectively known as inflammatory bowel diseases (IBD), are caused in part by overly-aggressive, T cell-mediated immune responses to bacterial products in genetically susceptible hosts. Isolates of Escherichia coli (E. coli) have been associated with Crohn's disease and bacterial components, including stress-response proteins, promote intestinal inflammation by stimulating innate and adaptive host immune systems. While the immune response to bacterial products has been extensively studied, little is known about the subsequent effects of host inflammation on bacterial properties. Selective colonization with a commensal murine strain of E. coli (NC101) causes colitis in IL-10-deficient (IL-10-/-), but not wild-type (wt), mice. Intestinal inflammation triggers NC101 to upregulate the bacterial stress-response gene ibpB. IbpB is a small heat shock protein that helps bacteria tolerate heat stress and high levels of reactive oxygen species (ROS) as is found in the inflamed intestine and macrophage phagolysosomes. HYPOTHESIS: The host inflammatory milieu upregulates bacterial ibpB, which in turn allows bacteria to adapt to the environment and perpetuate inflammation by increasing their survival and virulence. SPECIFIC AIMS: 1) Identify mechanisms by which the host inflammatory milieu stimulates E. coli to upregulate ibpB in vitro. We will study the roles of intracellular and secreted factors in upregulating E. coli ibpB using ibpB-eGFP reporter bacteria and inhibitors of ROS and cytokine production. 2) Ascertain the effects of E. coli ibpB expression on host inflammatory pathways in vitro. We will determine mechanisms by which E. coli IbpB inhibits uptake by macrophages and promotes intracellular survival using genetically altered NC101 and inhibitors of phagocytosis and ROS generation in gentamicin protection assays. 3) Examine the biological relevance of E. coli IbpB in the initiation and perpetuation of chronic experimental colitis in E. coli monoassociated IL-10-/- mice. We will measure histological inflammation, bacterial distribution, innate and adaptive immune responses in wt and IL-10-/- mice monoassociated with NC101 or ibpB-deficient NC101. CAREER DEVELOPMENT: Long-term goals are to lead a research team at an academic institution to: 1) contribute new knowledge to the field of host-microbial interactions, and 2) develop novel diagnostic strategies and therapies for IBD. Short-term goals are to: 1) learn new techniques to complete the proposed and future studies, 2) interact regularly with an exceptional mentoring team, 3) participate in didactic courses and research seminars in microbial genetics and pathogenesis, and 4) differentiate myself from my mentor by publishing manuscripts and submitting applications for independent funding in the area of bacterial stress responses. ENVIRONMENT: The National Gnotobiotic Rodent Resource Center, the Center for Gastrointestinal Biology and Disease, local experts in mucosal immunology and bacteriology, university start-up funds, and an institutional commitment to protect research time will facilitate attainment of these goals. PUBLIC HEALTH RELEVANCE: Inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, are chronic intestinal inflammatory disorders that cause significant morbidity. The studies in this proposal are designed to investigate the mechanisms by which resident intestinal bacteria may cause these diseases and to identify potential targets for novel therapies.