The Role of Reactive Oxygen Species in Mucosal Innate Immunity

描述（由申请人提供）：了解粘膜表面的先天免疫对于预防这些表面可能发生的传染病因子和炎症性免疫病理至关重要。一个鲜为人知的先天免疫机制是由粘膜组织中的双氧化酶产生和调节活性氧（ROS）的产生。本应用程序的目的是确定ROS产生的成分和防止自我伤害的机制。秀丽隐杆线虫将被使用，因为它是一种可接近的模式生物，我们可以用它来解决这些问题。核心假设是，在感染部位，肠细胞通过Ce-Duox1产生细胞外ROS，同时产生抗氧化剂和热休克蛋白，以防止自我损伤。提出这项研究的基本原理是，对秀丽隐杆线虫中ROS产生的成分和机制的了解可能适用于更复杂的动物，从而进一步了解ROS在粘膜先天免疫中的作用。目的1将确定Ce-Duox1的定位及其在应对病原体时产生ROS的活性。基于Ce-Duox1在肠道中产生ROS的工作假设，该酶将通过免疫荧光和gfp标记技术定位到该感染部位。通过使用对活性氧敏感的染料，这些物种也将被定位到感染部位。目标2将确定参与活性氧产生的调控机制和其他辅助因子。我们已经建立了一种检测方法，可以检测秀丽隐杆线虫对病原体的反应中ROS的产生。使用RNAi和突变体来检查特定基因的丢失，我们将研究p38 MAPK通路的参与，这在我们的初步研究中已经涉及到。其他已建立的免疫途径也将被研究。除了这些靶向方法外，还将对表现出ROS产生变化的突变体进行正向遗传筛选。在Aim #3中，我们将研究宿主如何将ROS造成的损害最小化。RNAi初步鉴定的抗氧化基因和热休克蛋白在感染过程中具有保护作用，将进一步分析。它们假定的保护作用将通过分析缺失突变体或过度表达感兴趣基因的转基因来证实。它们将通过gfp标记技术定位到感染部位。最后，通过检测脂褐素积累和蛋白质聚集，将评估这些基因对ros相关损伤的影响。综上所述，秀丽隐杆线虫将作为一个模型系统来回答关于ROS在粘膜免疫反应中的一些重要问题。作为拟议调查的结果，这种反应的组成部分、监管机构和损害控制机制将被确定和定位。这项研究具有重要意义，因为了解粘膜产生的ROS将有可能为调节这种免疫反应提供新的方法，从而治疗这些组织中的传染病和炎症。

DESCRIPTION (provided by applicant): Understanding the innate immunity of the mucosal surfaces is crucial to protecting against infectious disease agents and inflammatory immune pathologies that can occur at these surfaces. One poorly understood innate immune mechanism is the generation and regulation of reactive oxygen species (ROS) production by dual oxidases in mucosal tissue. The objective of this application is to identify the components of ROS production and the mechanisms that prevent self-damage. C. elegans will be used because it is an accessible model organism with which we can address these questions. The central hypothesis is that at the site of infection, the intestinal cells generate extracellular ROS via Ce-Duox1 while simultaneously producing antioxidants and heat shock proteins to prevent self-damage. The rationale for the proposed research is that knowledge of the components and mechanisms involved in ROS production in C. elegans will likely be applicable to more complex animals and therefore further understanding of ROS's role in mucosal innate immunity. Aim #1 will establish the localization of Ce-Duox1 and its ROS generating activity in response to pathogens. Based on the working hypothesis that Ce-Duox1 generates ROS in the intestine, the enzyme will be localized to this site of infection by immunofluorescence and GFP-tagging techniques. By using dyes sensitive to ROS, these species will also be localized to the site of infection. Aim #2 will identify regulatory mechanisms and other co-factors involved in ROS production. We have established an assay in which we can detect ROS production from C. elegans in response to pathogens. Using RNAi and mutants to examine the loss of specific genes we will investigate the involvement of the p38 MAPK pathway, which has been implicated in our preliminary studies. Other established immune pathways will also be investigated. In addition to these targeted approaches, a forward-genetic screen will be carried out for mutants that exhibit changes in ROS production. In Aim #3, we will investigate how the host minimizes damage caused by ROS. Antioxidant genes and heat shock proteins identified in preliminary studies by RNAi as having protective roles during infection will be further analyzed. Their putative protective roles will be confirmed by analyzing deletion mutants or transgenics that overexpress the gene-of-interest. They will be localized by GFP-tagging techniques to the site of infection. Finally, by examining lipofuscin accumulation and protein aggregation, these genes' effects on ROS-related damage will be assessed. In conclusion, C. elegans will be used as a model system to answer some important questions about ROS in mucosal immune response. As a result of the proposed investigations the components, regulators, and damage-controlling mechanisms of this response will be identified and localized. The research proposed is significant because knowledge of ROS production by the mucosa will potentially lead to new approaches for modulating this immune response in the treatment of infectious disease and inflammatory conditions in these tissues. PUBLIC HEALTH RELEVANCE: The research proposed in this application will lead to greater understanding of how an immune mechanism associated with mucosal surfaces, as found in the gastrointestinal and respiratory tracts, works. Specifically, using a tiny worm called C. elegans as a model, the mechanisms that generate, regulate and prevent self- damage from the response will be identified. Such knowledge is relevant to public health because it will lead to the potential manipulation of this immune response to the patient's advantage in the treatment of infections and autoimmune disorders associated with the mucosa.