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.