GM-CSF Regulation of Zinc in Histoplasmosis

描述(申请人提供)：二相性真菌，组织胞浆菌(HC)，是美国中西部和东南部特有的，是最常见的真菌呼吸道感染的原因。生物体在巨噬细胞的细胞内环境中茁壮成长，并建立一种潜伏状态。利用正向金属组学方法，我们的研究表明，巨噬细胞被粒细胞巨噬细胞集落刺激因子(GM-CSF)激活后，细胞内锌含量急剧减少，以应对HC感染。锌的缺乏与锌结合物种的显著增加有关。我们还发现，白介素4可以恢复细胞内的生长，同时增加细胞内的锌。因此，我们收集了大量数据，表明限制锌的获取是GM-CSF激活阻止HC细胞内生长的主要机制。这一新的发现使我们推测锌限制可能是细胞因子施加的一种重要的寄主抗性机制。在此，我们提出了三个具体目标。第一个目标是确定人巨噬细胞激活GM-CSF是否会降低宿主细胞和HC中的锌含量。在第二个目标中，我们将定量和鉴定人和小鼠巨噬细胞中上调的锌结合蛋白，并选择通过siRNA沉默的候选蛋白来确定它们在锌调节中的作用。目的3将检测缺乏GM-CSF的小鼠巨噬细胞群中的锌含量和锌结合种类。这个探索性的建议建立在一个新的和令人兴奋的观察结果的基础上，即细胞因子如何激活巨噬细胞来表达抗真菌和可能的抗微生物活性。我们的工作将努力确定这种微量金属在宿主与微生物的战斗中的必要性。 与公共卫生相关：致病真菌，组织胞浆菌，在巨噬细胞中繁殖，直到它们被细胞因子激活。被刺激的巨噬细胞对酵母的杀灭是由锌饥饿所介导的。我们试图确定锌枯竭是如何发生的，以便这些发现可以为这种真菌的治疗提供新的途径。

DESCRIPTION (provided by applicant): The dimorphic fungus, Histoplasma capsulatum (Hc), is endemic to the midwestern and southeastern United States and is the most frequent cause of fungal respiratory infections. The organism thrives within the intracellular environment of macrophages and establishes a latent state. Using a forward metallomics approach, our studies indicate that activation of macrophages by granulocyte macrophage colony-stimulating factor (GM-CSF) sharply limits intracellular zinc content in response to infection with Hc. The deprivation of zinc is associated with marked increases in zinc binding species. We also have shown that interleukin-4 restores intracellular growth and concomitantly increases intracellular zinc. We therefore have gathered substantial data to indicate that limiting access to zinc is a major mechanism by which GM-CSF activation halts intracellular growth of Hc. This novel finding has led us to hypothesize that zinc limitation may be an important host resistance mechanism exerted by cytokines. Herein, we propose 3 specific aims. The first aim is to determine if GM-CSF activation of human macrophages decreases zinc content in host cells and Hc. In the second aim, we will quantify and identify the upregulated zinc binding proteins in both human and mouse macrophages and select candidates to silence by siRNA to determine their role in zinc regulation. Aim 3 will examine zinc content and zinc binding species among macrophage populations in mice in which GM-CSF is absent. This exploratory proposal builds upon a new and exciting observation regarding how cytokines may activate macrophages to express antifungal and perhaps antimicrobial activity. Our work will endeavor to establish the necessity of this trace metal in the host-microbe battle. PUBLIC HEALTH RELEVANCE: The pathogenic fungus, Histoplasma capsulatum, multiplies in macrophages until they are activated by cytokines. Killing of yeasts by stimulated macrophages is mediated by zinc starvation. We seek to determine how zinc depletion occurs so that these findings can offer new avenues for treatment of this fungus.