The role of reactive oxygen species in mucosal innate immunity

 描述（由申请人提供）：双氧化酶（DUOX）产生的过氧化氢（H2 O2）如何有助于粘膜表面防御感染尚不完整，这是一个关键的知识空白。本研究的长期目标是阐明活性氧（ROS）如何在上皮组织中促进先天免疫。本申请的总体目标是使用C.优雅的模特中心假设是BLI-3（C. elegans DUOX）有助于免疫防御（1）作为影响氧化还原状态的信号分子，从而影响细胞保护性转录因子的活性，和（2）作为免疫特异性过氧化物酶的底物。该项目的基本原理是，更全面地了解DUOX酶在感染期间如何产生H2 O2，从而有助于免疫应答，这将可能有助于其治疗调节。 中心假设将通过以下目标来解决。具体目标1：确定硫氧还蛋白TRX-1如何调节保护性转录因子SKN-1。我们将检验工作假设，即在还原条件下，TRX-1通过直接结合使SKN-1调节因子NSY-1失活。在氧化条件下，例如在免疫应答期间发生的那些条件下，TRX-1中氧化还原敏感性半胱氨酸的氧化释放NSY-1。用于检验这一假设的方法将包括研究trx-1和nsy-1的适当突变体以及体内和体外方法来检验TRX-1和NSY-1之间的直接相互作用。具体目标2：阐明假定的过氧化物酶促进病原体抗性的机制。我们已经确定了假定的过氧化物酶，有助于免疫防御，我们将测试的假设，他们这样做，利用过氧化氢，以产生更强大的氧化剂。用于检验这一假设的方法将包括对具有这些基因突变版本的动物进行表征，以及对它们编码的蛋白质进行纯化和酶分析。具体目标3：确定BLI-3是否与推定的过氧化物酶直接相互作用。我们的工作假设是，这些过氧化物酶直接与BLI-3相互作用，BLI-3是产生其底物的酶。用于测试这一假设的方法将包括通过遗传和细胞生物学技术以及体内和体外方法来测试与BLI-3的直接相互作用的过氧化物酶的定位。这项研究具有重要意义，因为了解双氧化酶产生的H2 O2如何影响生物系统可能会导致调节其积极和消极影响的治疗。例如，不断增加ROS的产生量可能会导致对感染性疾病的保护，而减少ROS可能有助于解决炎症状况。该研究具有创新性，因为它利用了C. elegans独特的功能，产生一个单一的DUOX酶，以了解DUOX产生的H2 O2在感染过程中的作用，在整个生物体的自然环境。无需考虑其他NADPH氧化酶的影响，将有助于在生物体水平上研究DUOX。

 DESCRIPTION (provided by applicant): How hydrogen peroxide (H2O2) produced by dual oxidases (DUOXs) contributes to the defense of mucosal surfaces against infection is incomplete, representing a critical gap in knowledge. The long-term goal of this re- search is to elucidate how ROS (reactive oxygen species) contribute to innate immunity in epithelial tissues. The overall objective of this application is to identify how H2O2 produced by DUOXs contributes to immune defense, using C. elegans as a model. The central hypothesis is that the H2O2 produced by BLI-3 (C. elegans DUOX) contributes to immune defense (1) as a signaling molecule that affects the redox state and therefore the activity of a cytoprotective transcription factor, and (2) as a substrate for immune-specific peroxidases. The rationale for the project is that a more complete understanding of how H2O2 production by DUOX enzymes during infection contributes to the immune response will potentially allow for its therapeutic modulation. The central hypothesis will be addressed by the following aims. Specific Aim 1: Determine how thioredoxin TRX-1 regulates the protective transcription factor SKN-1. We will test the working hypothesis that under reducing conditions TRX-1 inactivates the SKN-1 regulator NSY-1, by direct binding. Under oxidizing conditions, such as those that occur during the immune response, oxidation of redox-sensitive cysteines in TRX-1 releases NSY-1. The approaches used to test this hypothesis will include studying appropriate mutants of trx-1 and nsy-1 and in vivo and in vitro approaches to test for a direct interaction between TRX-1 and NSY-1. Specific Aim 2: Elucidate the mechanism(s) by which putative peroxidases contribute to pathogen resistance. We have identified putative peroxidases that contribute to immune defense, and we will test the postulate that they do so by utilizing H2O2 to generate more powerful oxidants. The approaches used to test the hypothesis will include characterization of animals with mutated versions of these genes and purification and enzymatic analysis of the proteins they encode. Specific Aim 3: Determine if BLI-3 directly interacts with the putative peroxidases. Our working hypothesis is that these peroxidases interact directly with BLI-3, the enzyme that produces their substrate. The approaches used to test this hypothesis will include localization of the peroxidases by genetic and cell-biological techniques and in vivo and in vitro approaches to test for a direct interaction with BLI-3. The research proposed is significant because knowledge of how the H2O2 produced by dual oxidases affects biological systems could lead to treatments that modulate its positive and negative effects. For example, pharmacologically increasing the amount of ROS production may lead to protection against infectious diseases, while decreasing ROS may help resolve inflammatory conditions. The proposed research is innovative be- cause it takes advantage of C. elegans unique feature of producing a single DUOX enzyme to understand the roles of DUOX-generated H2O2 during infection in the natural context of the whole organism. Not having to ac- count for the effects of other NADPH oxidases will facilitate the study of DUOX at the organismal level.