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），部分由遗传易感宿主对细菌产物的过度攻击性T细胞介导的免疫应答引起。分离的大肠杆菌（E.大肠杆菌）与克罗恩病和细菌成分，包括应激反应蛋白，通过刺激先天和适应性宿主免疫系统促进肠道炎症。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。选择性定殖与E.大肠杆菌（NC 101）在IL-10缺陷型（IL-10-/-）小鼠中引起结肠炎，但在野生型（wt）小鼠中不引起结肠炎。肠道炎症触发NC 101上调细菌应激反应基因ibpB。IbpB是一种小的热休克蛋白，有助于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：宿主炎症环境上调细菌ibpB，这反过来又允许细菌适应环境，并通过增加其存活率和毒力使炎症持续存在。具体目的：1）确定宿主炎症环境刺激E。coli中诱导ibpB表达。我们将研究细胞内和分泌因子在上调E.使用ibpB-eGFP报告细菌和ROS和细胞因子产生的抑制剂，2)确定E. coli ibpB表达对宿主炎症通路的影响。我们将确定的机制，E。在庆大霉素保护试验中，使用遗传改变的NC 101和吞噬作用和ROS产生的抑制剂，coli IbpB抑制巨噬细胞的摄取并促进细胞内存活。3)检测E. coli IbpB在慢性实验性结肠炎的发生和延续中的作用。coli单相关IL-10-/-小鼠。我们将测量与NC 101或ibpB缺陷型NC 101单相关的野生型和IL-10-/-小鼠中的组织学炎症、细菌分布、先天性和适应性免疫应答。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IBD的新诊断策略和疗法。短期目标是：1）学习新技术，以完成拟议的和未来的研究，2）与一个特殊的指导团队定期互动，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，和4）通过发表手稿和提交申请，使自己与我的导师在细菌应激反应领域的独立资金区分开来。环境：国家非生物啮齿类动物资源中心、胃肠生物学和疾病中心、当地粘膜免疫学和细菌学专家、大学启动资金以及保护研究时间的机构承诺将有助于实现这些目标。 公共卫生相关性：炎症性肠病，包括克罗恩病和溃疡性结肠炎，是引起显著发病率的慢性肠道炎症性疾病。该提案中的研究旨在调查肠道细菌可能导致这些疾病的机制，并确定新疗法的潜在靶点。

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.