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性免疫病理至关重要。一个知之甚少的先天免疫机制是粘膜组织中双氧化酶产生和调节活性氧（ROS）的产生。本申请的目的是确定ROS产生的组分和防止自我损伤的机制。C.我们将使用秀丽线虫，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，了解C。elegans将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-Duox 1的定位及其响应病原体的ROS生成活性。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标记技术定位于该感染部位。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将确定参与ROS产生的调节机制和其他辅助因子。我们建立了一种检测C.线虫对病原体的反应使用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被牵连。还将研究其他已建立的免疫途径。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，我们将研究宿主如何最大限度地减少ROS造成的损害。在初步研究中通过RNAi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。通过分析过表达目标基因的缺失突变体或转基因体，将证实它们的推定保护作用。它们将通过GFP标记技术定位于感染部位。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。结论是C. elegans将作为一个模型系统，以回答一些重要的问题，活性氧在粘膜免疫反应。作为拟议的调查的结果，组件，监管机构和损害控制机制，这种反应将被确定和本地化。提出的研究是重要的，因为粘膜产生ROS的知识将可能导致在这些组织中治疗感染性疾病和炎症性疾病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使人们更好地理解与胃肠道和呼吸道中发现的粘膜表面相关的免疫机制如何发挥作用。具体来说，使用一种名为C的微小蠕虫。以秀丽隐杆线虫为模型，研究其产生，调节和防止自我损伤的机制。这样的知识与公共卫生相关，因为它将导致对这种免疫应答的潜在操纵，从而在治疗与粘膜相关的感染和自身免疫性疾病中对患者有利。

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.