Project Summary Fungal infection with Histoplasma species (Hc) is highly prevalent in the Ohio and Mississippi river valley regions of the US and is a frequent cause of fungal respiratory illness. Macrophages are the frontline effector cells that engulf Hc but only kill yeast cells upon activation. Emerging evidence indicates that macrophages and other innate cells develop a “memory” phenotype by remembering a prior microbial encounter. This process has been referred to as trained immunity. Trained macrophages manifest heightened antimicrobial defenses independent of T and B cells. Thus, training represents an alternative for improving the outcome of infection in patients with AIDS or other immunocompromising conditions. Metabolic changes, in particular, elevated glycolysis and de- creased oxidative phosphorylation are key to the development of trained macrophages. The precise mechanisms underlying training have not been fully uncovered, and the effect of trained macrophages on Hc resistance is unknown. We have generated exciting data that training human and mouse macrophages enables them to elim- inate infection with yeast cells independent of exogenous cytokine activation. Using molecular, immunologic, and metallomic approaches, we discovered that the labile zinc content of macrophages is diminished during training concomitant with an elevated expression of the zinc binding proteins metallothioneins (MTs)1&2. Trained macrophages inhibit Hc growth in a zinc-dependent manner. How the MT-zinc axis influences the development and execution of memory in macrophages is not understood. In this proposal, we will investigate the hypothesis that zinc homeostasis is a central element in sculpting macrophage memory. Aim 1 will explore the role of gran- ulocyte macrophage-colony stimulating factor and low zinc in the development of memory. We chose to study this cytokine since many of its effects on zinc mirror that of training. Aim 2 will determine how MTs1&2 orchestrate macrophage training. Aim 3 will decipher how the MT-zinc axis regulates glycolytic reprogramming and investi- gate how metabolic changes shape the anti-Hc response of trained macrophages. Collectively, these studies will provide new knowledge regarding zinc regulation of trained immunity.