The Role of Reactive Oxygen Species in Mucosal Innate Immunity

描述(由申请人提供)：了解粘膜表面的天然免疫力对于预防这些表面可能发生的感染性疾病和炎性免疫病理至关重要。一种鲜为人知的先天免疫机制是通过粘膜组织中的双重氧化酶产生和调节活性氧物种(ROS)的产生。这个应用程序的目标是确定ROS产生的成分和防止自我损害的机制。线虫将被使用，因为它是一种可访问的模式生物，我们可以用它来解决这些问题。中心假设是，在感染部位，肠道细胞通过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身损害。这项拟议研究的基本原理是，对线虫体内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建立Ce-Duox1的定位及其在病原体刺激下产生ROS的活性。根据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ROS敏感的染料，这些物种也将定位于感染部位。目标2将确定调节机制和其他参与ROS生产的辅助因素。我们已经建立了一种检测方法，可以检测线虫对病原体的反应产生ROS。使用RNAi和突变体来检测特定基因的丢失，我们将调查p38MAPK通路的参与，这已经在我们的初步研究中涉及。其他已建立的免疫途径也将受到调查。除了这些有针对性的方法外，还将对表现出ROS产生变化的突变体进行正向遗传筛选。在目标3中，我们将调查主机如何将ROS造成的损害降至最低。RNAi初步研究中确定的在感染期间具有保护作用的抗氧化基因和热休克蛋白将被进一步分析。它们假定的保护作用将通过分析过度表达感兴趣基因的缺失突变体或转基因来证实。它们将通过GFP标签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，将评估这些基因在ROS相关损伤中的作用。综上所述，线虫将作为一个模型系统来回答有关粘膜免疫应答中ROS的一些重要问题。作为拟议调查的结果，将确定这一反应的组成部分、监管机构和损害控制机制，并将其本地化。这项研究具有重要意义，因为了解粘膜产生ROS的知识可能会导致在治疗这些组织中的传染病和炎症情况时调节这种免疫反应的新方法。 公共卫生相关性：本申请中提出的研究将有助于更好地理解胃肠道和呼吸道中发现的与粘膜表面相关的免疫机制是如何工作的。具体地说，以一种名为线虫的微小蠕虫为模型，将识别产生、调节和防止反应中自我损害的机制。这种知识与公共健康相关，因为它将导致潜在的对这种免疫反应的操纵，使患者在治疗与粘膜相关的感染和自身免疫性疾病方面具有优势。

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.