L-citrulline and anti-tuberculosis host defense

项目摘要/摘要 目前的抗结核(TB)战略需要数月的治疗，而开发多种药物再治疗 结核分支杆菌耐药突变株(Mtb)增加了治疗的复杂性和成本。因此， NIAID最近建议以感染者对感染的反应为目标，以此作为加强抗结核病的手段 防御。这些“宿主导向疗法”可以与已被证实的抗结核抗生素策略相结合， 全面加强治疗和病人护理。氨基酸是免疫功能不可或缺的一部分， 然而，在理解靶向氨基酸代谢的治疗潜力方面存在着根本性的差距。 在疾病期间。长期目标是确定氨基酸代谢和免疫反应之间的相互作用。 海绵，为控制免疫活动提供了新的治疗途径。这项研究的目的是 确定L-瓜氨酸代谢在巨噬细胞(M-Ф)介导的结核分枝杆菌免疫应答中的作用。美联社- Plicant将用结核分枝杆菌H37Rv感染人和小鼠的MФS，以及体内感染的小鼠进行检测 L-结核分枝杆菌感染期间的瓜氨酸代谢。中心假设是L-瓜氨酸代谢是必需的 具有抗结核M-Ф活性，可用于体内辅助宿主对结核的防御。假设是支持的-- ED作为a)L-瓜氨酸在体外促进MΦNO的产生和抗结核分枝杆菌的活性，b)L-瓜氨酸通过 髓样细胞是体内分枝杆菌防御所必需的，c)L-瓜氨酸补充减少肺 分枝杆菌负荷和d)肺MФS是主要的L-瓜氨酸在感染期间利用细胞。Ra- 这项拟议的研究的理由是，揭示免疫介导的感染控制机制很可能 导致治疗结核分枝杆菌感染者的新方法-每年导致100多万人死亡。应用程序- 坎特将通过调查三个具体目标来检验中心假设：1)检验L-瓜氨酸是如何利用- 在结核分枝杆菌感染的MФS体内，2)确定L-瓜氨酸在感染结核杆菌的人MΦS中的代谢，以及3)确定L-瓜氨酸在结核分枝杆菌感染的人M 确定利用L-瓜氨酸代谢如何增强抗分枝杆菌宿主防御。在第一个下 第二个目标是，申请人将利用创新的细胞培养和质谱学AP-AP的组合。 用氨基酸浓度滴定法测定L-瓜氨酸代谢对沪猪的效益(S) 人和小鼠MФS。这些实验将定义这一途径的机械后果 补充第三个目标下的活体实验，其中申请人将使用原创方法来增强- L瓜氨酸对小鼠肺内Hance Mtb的清除作用。这项拟议的研究具有重要意义 正如我们预期的那样，利用L-瓜氨酸代谢将增强M-Ф介导的对结核病的控制，并且结合- 使用抗分枝杆菌抗生素治疗将为患者提供有效的治疗策略 得了肺结核。这项研究也有望对增强宿主防御机制产生广泛的影响 通过改变氨基酸代谢来控制病原体和免疫系统的个别成分。

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.