Bacterial Stress Responses to Intestinal Inflammation

描述(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略性T细胞介导的免疫反应引起的。大肠杆菌的分离株与克罗恩病有关，包括应激反应蛋白在内的细菌成分通过刺激天然和适应性宿主免疫系统促进肠道炎症。虽然对细菌产品的免疫反应已经进行了广泛的研究，但对宿主炎症对细菌特性的后续影响知之甚少。共栖小鼠大肠杆菌(NC101)的选择性定植引起IL-10缺陷(IL-10-/-)小鼠的结肠炎，但不引起野生型(Wt)小鼠的结肠炎。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，可以帮助细菌耐受热应激和高水平的活性氧物种(ROS)，就像在发炎的肠道和巨噬细胞吞噬酶体中发现的那样。假设：宿主炎症环境上调细菌ibpB，进而允许细菌适应环境，并通过增加它们的存活率和毒力来维持炎症。具体目的：1)确定宿主炎症环境在体外刺激大肠杆菌表达ibpB的机制。我们将利用ibpB-EGFP报告细菌和ROS和细胞因子产生的抑制剂来研究细胞内和分泌因素在上调大肠杆菌ibpB中的作用。2)确定大肠杆菌ibpB在体外表达对宿主炎症途径的影响。我们将使用基因改变的NC101和庆大霉素保护试验中的吞噬和ROS生成抑制剂来确定大肠杆菌IbpB抑制巨噬细胞摄取和促进细胞内存活的机制。3)检测大肠杆菌IbpB在IL-10-/-小鼠慢性实验性结肠炎发病和持续发展过程中的生物学相关性。我们将测量与NC101或ibpB缺陷NC101单一相关的wt和IL-10-/-小鼠的组织炎症、细菌分布、先天性和获得性免疫反应。职业发展：长期目标是领导一个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的新诊断策略和治疗方法。短期目标是：1)学习新技术以完成拟议和未来的研究，2)定期与杰出的指导团队互动，3)参加微生物遗传学和发病机制的教学课程和研究研讨会，4)通过出版手稿和提交细菌应激反应领域的独立资金申请，使自己与我的导师区分开来。环境：国家生态型啮齿动物资源中心、胃肠道生物学和疾病中心、当地黏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促进这些目标的实现。 与公共卫生相关：炎症性肠道疾病，包括克罗恩病和溃疡性结肠炎，是一种慢性肠道炎症性疾病，会导致显著的发病率。这项建议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.