Modulating Nrf2-Reguated GSH Production to Prevent Hospital-Acquired Infections

描述(申请人提供)：医院获得性感染是重症监护病房死亡和发病的主要原因，由于免疫抑制，危重患者特别容易受到细菌感染。由于越来越多的医院获得性感染是由耐多药生物引起的，因此需要新的治疗方法来预防和治疗重症监护病房的细菌感染。我们的长期目标是开发一种有效的治疗策略，通过增强中性粒细胞的杀菌活性来克服危重患者的免疫抑制。危重病患者经常表现出氧化应激增加，细胞内主要抗氧化剂谷胱甘肽(GSH)水平下降。谷胱甘肽的生成有两条途径：氧化谷胱甘肽的从头合成和再生。我们实验室最近的研究表明，缺乏谷胱甘肽还原酶(GSR)的小鼠非常容易受到与医院获得性感染相关的几种细菌的感染，表现为高死亡率、严重的器官损伤和炎症加剧。对细菌感染易感性增加的根本原因是未能杀死感染的有机体。在没有GSR的情况下，中性粒细胞--负责根除细菌的主要白细胞--无法有效地摄取和杀死感染细菌，这可能是由于中性粒细胞过度氧化损伤所致。 他们自己。我们的研究表明，GSH再生是中性粒细胞介导的宿主防御的关键，这引发了一个有趣的问题，即过度氧化应激本身是否导致危重患者出现免疫抑制。我们的发现有力地表明，从头合成GSH对于有效抵抗细菌病原体的免疫防御至关重要，因为GSR的活性取决于从头合成GSH，即没有GSH就不会有GSSG。谷氨酸-半胱氨酸连接酶是谷胱甘肽从头合成的限速步骤，它由催化亚基(GCLC)和调节亚基(GCLM)组成。转录因子Nrf2调节GSR、Gclm和Gclc的表达，从而调节GSSG的从头合成和GSH再生。我们建议的中心假设是，NRF2调节的GSH抗氧化系统通过维持中性粒细胞的杀菌活性来促进宿主防御。这项建议的目标是 确定GSH在中性粒细胞介导的宿主防御中的作用，并评估Nrf2激活作为增强宿主防御的方法的有效性。R21建议的具体目的是确定两条GSH生成途径在抗细菌免疫防御中的功能(目标1)，并评估Nrf2激活作为一种增强中性粒细胞杀菌活性的治疗策略的有效性(目标2)。我们的研究将阐明危重患者免疫抑制的潜在机制。我们的研究还将促进新型治疗药物的开发，用于预防和治疗重症监护病房的医院获得性感染。

DESCRIPTION (provided by applicant): Hospital-acquired infections are a major cause of mortality and morbidity in intensive care units, and critically ill patients are particularly susceptible to bacterial infections due to immunosuppression. Since an increasingly greater portion of the hospital-acquired infections are caused by multidrug-resistant organisms, new therapeutic approaches are needed to prevent and treat bacterial infections in intensive care units. Our long- term goal is to develop an effective therapeutic strategy to overcome immunosuppression in critically ill patients through bolstering the bactericidal activity of neutrophils. Critically ill patients often display increased oxidative stress and have decreased levels of glutathione (GSH), a major intracellular antioxidant. GSH is generated through two pathways: de novo synthesis and regeneration from oxidized glutathione (GSSG). Recent studies in our laboratory demonstrated that mice lacking glutathione reductase (Gsr), which catalyzes GSH regeneration from GSSG, are highly vulnerable to infection by several bacterial species associated with hospital-acquired infections, as indicated by high rates of death, severe organ damage, and enhanced inflammation. The underlying cause of the increased vulnerability to bacterial infection is the failure to kill the infecting organism. In the absence of Gsr, neutrophils, the primary white blood cells responsible for bacterial eradication, cannot efficientl ingest and kill the infecting bacteria, likely due to excessive oxidative damage to the neutrophils themselves. Our studies indicate that GSH regeneration is pivotal for neutrophil-mediated host defense, raising the intriguing question of whether excessive oxidative stress is itself responsible for the immunosuppression seen in critically ill patients. Our findings strongly suggest that de novo GSH synthesis will be crucial for an effective immune defense against bacterial pathogens, since Gsr activity depends on de novo GSH synthesis, i.e. without GSH there can be no GSSG. The rate-limiting step in de novo GSH synthesis is glutamate-cysteine ligase, which consists of a catalytic subunit (Gclc) and a modulatory subunit (Gclm). The transcription factor Nrf2 regulates the expression of Gsr, Gclm, and Gclc, and thus Nrf2 modulates both de novo GSH synthesis and GSH regeneration from GSSG. The central hypothesis of our proposal is that the GSH antioxidant system modulated by Nrf2 facilitates host defense by maintaining bactericidal activity in neutrophils. The objectives of this proposal are to determine the role of GSH in neutrophil-mediated host defense and to assess the efficacy of Nrf2 activation as an approach to enhance host defense. The specific aims of this R21 proposal are to define the functions of the two GSH-generating pathways in anti-bacterial immune defense (Aim 1), and to evaluate the efficacy of Nrf2 activation as a therapeutic strategy to bolster neutrophil bactericidal activity (Aim 2). Our studies will elucidate a potential mechanism underlying immunosuppression in critically ill patients. Our studies will also facilitate the development of novel therapeutic drugs for the prevention and treatment of hospital-acquired infections in intensive care units.