The Warburg effect and host immune response in tuberculosis

摘要 结核分枝杆菌（Mtb）是最成功的人类病原体，2015年造成180万人死亡。 越来越多的证据表明，结核分枝杆菌的生存能力，持续存在和导致疾病的主要原因是它的 破坏宿主对感染的免疫和抗菌反应的能力。的最新进展 免疫代谢已经表明，代谢向糖酵解的转变，也就是瓦尔堡效应，对于 淋巴细胞的活化和分化，树突状细胞成熟，以及M1巨噬细胞极化， 其与微生物杀灭和感染的有效控制相关。然而，人们对此知之甚少。 结核分枝杆菌感染过程中免疫细胞的代谢状态及其在结核病发病机制中的作用。使用转录组学和 通过荧光免疫组化辅助成像，我们发现代谢重构的证据与瓦尔堡 在巨噬细胞离体Mtb感染期间以及在小鼠、兔和人中Mtb感染期间的作用 肺更有趣的是，我们观察到肉芽肿中心的感染巨噬细胞显示出减少， 瓦尔堡效应状态与外周的相比，表明Mtb干扰宿主细胞的代谢开关 以削弱其促炎和抗菌功能。基于这些和文献中的其他数据，我们 假设Mtb干扰瓦尔堡效应以抑制APC极化和功能， 炎症和抗菌功能的适应性免疫和抑制巨噬细胞活化，因此， 有利于病原体的存活和持续。为了验证我们的假设，我们提出了三个具体目标。 首先，我们将确定瓦尔堡效应状态与巨噬细胞极化、活化 和T细胞分化在肉芽肿的兔模型肺活动性结核和潜伏感染，使用 基于荧光IHC和单分子RNA-FISH（smFISH）的成像。我们还将进行代谢组学 在分化和成熟的不同阶段的肉芽肿区域的分析。第二，我们将扰乱 商业验证的治疗性小分子化合物和SiRNA敲除的瓦尔堡效应 并分析这种扰动对先天性和适应性免疫细胞的效应器功能的影响 以及在体内肺结核的小鼠模型中。第三，我们将使用RNA-Seq和基于IHC和smFISH的成像技术， 分析代谢/瓦尔堡效应决定因素，这些决定因素负责潜伏期的建立和 重新激活我们还将描述小分子治疗剂对瓦尔堡效应扰动的影响。 在兔潜伏期模型中观察化合物对宿主免疫应答和Mtb生长动力学的影响。通过阐明 瓦尔堡效应与宿主先天免疫和适应性免疫功能潜力之间的相关性 结核病及其与感染结果的关系，这项研究将建立对结核病的一个新方面的理解 致病性这项研究的结果可能会导致宿主导向疗法的发展，以靶向 免疫细胞的代谢，以增强其抗菌反应，促进努力控制和消除 这种致命的疾病！

Abstract Mycobacterium tuberculosis (Mtb) is the most successful human pathogen, causing 1.8 million deaths in 2015. Accumulating evidence suggests that Mtb’s ability to survive, persist and cause disease is largely due to its ability to subvert the host immune and antimicrobial response to infection. Recent advances in immunometabolism have shown that a metabolic shift to glycolysis, aka the Warburg effect, is critical for the activation and differentiation of lymphocytes, dendritic cell maturation, and for M1 macrophage polarization, which is associated with microbial killing and effective control of infection. However, little is known about the metabolic state of immune cells during Mtb infection and its role in TB pathogenesis. Using transcriptomics and fluorescent IHC-assisted imaging, we found evidence for metabolic remodeling consistent with the Warburg effect during Mtb infection of macrophages ex vivo, as well as during Mtb infection in mouse, rabbit and human lungs. More intriguingly, we observed that infected macrophages at the center of granulomas showed decreased Warburg effect state compared to those at the periphery, suggesting that Mtb perturbs host cell metabolic switch to impair their pro-inflammatory and antimicrobial functions. Based on these and other data in the literature, we hypothesize that Mtb perturbs the Warburg effect to dampen APC polarization and function, compromising pro- inflammatory and antimicrobial functions of adaptive immunity and dampening macrophage activation, thus favoring the survival and persistence of the pathogen. To test our hypothesis, we propose three Specific Aims. First, we will determine the correlation between the Warburg effect state and macrophage polarization, activation and differentiation of T cells in granulomas in rabbit models of pulmonary active TB and latent infection, using fluorescent IHC- and single molecule RNA-FISH (smFISH)-based imaging. We will also perform metabolomic analysis of regions of granulomas at different stages of the differentiation and maturation. Second, we will perturb the Warburg effect by commercially validated therapeutic small molecule compounds and siRNA knockdown and analyze the effects of this perturbation on the effector functions of innate and adaptive immune cells ex vivo and in a mouse model of pulmonary TB in vivo. Third, we will use RNA-Seq and IHC- and smFISH-based imaging to dissect the metabolic/Warburg effect determinants responsible for the establishment of latency and for the reactivation. We will also characterize the effects of Warburg effect perturbation by small molecule therapeutic compounds on the host immune response and Mtb growth dynamics in a rabbit latency model. By elucidating the correlation between the Warburg effect and the functional potential of host innate and adaptive immunity in TB and its association with infection outcome, this study will establish an understanding of a novel aspect of Mtb pathogenicity. Outcomes of this study may lead to the development of host-directed therapies to target metabolism of immune cells to enhance their antimicrobial responses, facilitating efforts to control and eradicate this deadly disease.!