L-citrulline and anti-tuberculosis host defense

项目总结/摘要 目前的抗结核病（TB）策略需要数月的治疗，而多药治疗的发展， M.结核病（Mtb）增加了治疗的复杂性和成本。因此， NIAID最近提出了针对感染个体对感染的反应作为加强抗结核病的一种手段 的防御合作.这些“针对宿主的疗法”可以与针对结核病的已证实的抗生素策略相结合， 提供治疗和病人护理的整体增强。氨基酸是免疫功能的组成部分， 然而，在理解靶向氨基酸代谢的治疗潜力方面存在根本性的差距， 在疾病期间。长期的目标是确定氨基酸代谢和免疫重建之间的相互作用， sponses，提供新的治疗途径来操纵免疫活性。本研究的目的是 确定L-瓜氨酸代谢对巨噬细胞（Mtb）介导的免疫应答的作用。该ap- 申请人将使用Mtb H37 Rv感染人和小鼠的Mtb H37 Rv，以及小鼠的体内感染， 结核分枝杆菌感染期间的L-瓜氨酸代谢。中心假设是L-瓜氨酸代谢是必需的 用于抗结核分枝杆菌活性，并可用于协助宿主体内防御结核病。假设是支持- 艾德：a）L-瓜氨酸在体外增强MΦ NO的产生和抗Mt B活性，B）L-瓜氨酸代谢， 骨髓细胞是体内分枝杆菌防御所必需的，c）L-瓜氨酸补充剂降低肺 分枝杆菌负荷，和d）肺M-型细胞是感染期间主要的L-瓜氨酸利用细胞。那个... 这项研究的意义在于，揭示免疫介导的感染控制机制将有可能 导致了治疗结核病感染者的新方法--结核病每年导致100多万人死亡。应用程序- cant将通过调查三个具体目标来检验中心假设：1）检查L-瓜氨酸是如何被利用的， 2）确定L-瓜氨酸在感染Mtb的人MΦ中的代谢，3） 确定利用L-瓜氨酸代谢如何增强抗分枝杆菌宿主防御。根据第一项 第二个目标，申请人将利用创新的细胞培养和质谱分析的组合， 通过滴定氨基酸浓度来确定L-瓜氨酸代谢对人类的益处， 人和老鼠的M型这些实验将确定这一途径的机械后果， 补充第三个目标下的体内实验，其中申请人将使用原始方法， 通过向小鼠补充L-瓜氨酸来增强肺中Mtb的清除。所提出的研究是有意义的 因为我们预计利用L-瓜氨酸代谢将增强M β介导的结核病控制， 与抗分枝杆菌抗生素治疗的结合将为患有结核病的患者提供有效的治疗策略。 肺结核这项研究也有望对宿主防御机制产生广泛的影响， 病原体控制和免疫系统的个别组成部分通过改变氨基酸代谢。

PROJECT SUMMARY/ABSTRACT Current anti-tuberculosis (TB) strategies require months of treatment, and the development of multi-drug re- sistant mutants of M. tuberculosis (Mtb) has increased both the complexity and cost of treatment. As such, the NIAID recently proposed targeting infected individuals’ responses to infection as a means to enhance anti-TB defenses. These “host-directed therapies” could be combined with proven antibiotic strategies against TB, providing an overall enhancement of treatment and patient care. Amino acids are integral to immune function, yet there is a fundamental gap in understanding the therapeutic potential of targeting amino acid metabolism during disease. The long-term goal is to define the interplay between amino acid metabolism and immune re- sponses, providing new therapeutic avenues to manipulate immune activity. The objective of this study is to identify the role of L-citrulline metabolism on macrophage (MФ)-mediated immune responses to Mtb. The ap- plicant will use Mtb H37Rv infection in human and mouse MФs, as well as in vivo infection in mice, to examine L-citrulline metabolism during Mtb infection. The central hypothesis is that L-citrulline metabolism is required for anti-TB MФ activity and can be harnessed to assist host defense to TB in vivo. The hypothesis is support- ed as a) L-citrulline enhances MΦ NO production and anti-Mtb activity in vitro, b) L-citrulline metabolism by myeloid cells is necessary for mycobacterial defenses in vivo, c) L-citrulline supplementation decreases lung mycobacterial burden, and d) lung MФs are the predominant L-citrulline utilizing cells during infection. The ra- tionale for the proposed research is that uncovering mechanics of immune-mediated infection control will likely lead to novel methods for treating those infected with Mtb – which kills well over 1 million annually. The appli- cant will test the central hypothesis by investigating three specific aims: 1) to examine how L-citrulline is uti- lized in Mtb-infected MФs, 2) to define the metabolism of L-citrulline in human MΦs infected with Mtb, and 3) to identify how harnessing L-citrulline metabolism enhances anti-mycobacterial host defenses. Under the first and second aims, the applicant will utilize a combination of innovative cell culture and mass spectrometry ap- proaches with titrating amino acid concentrations to determine the benefit(s) of L-citrulline metabolism in hu- man and mouse MФs. These experiments will define the mechanistic consequences of this pathway that will complement in vivo experiments under the third aim, where the applicant will use an original approach to en- hance Mtb clearance in the lungs by supplementing mice with L-citrulline. The proposed research is significant as we anticipate harnessing L-citrulline metabolism will augment MФ-mediated control of TB, and in combina- tion with anti-mycobacterial antibiotic therapy will result in efficient treatment strategies for patients suffering with TB. This research is also expected to have broad implications on host defense mechanisms – enhancing pathogen control and individual components of the immune system by altering amino acid metabolism.