Fighting Histoplasma Capsulatum Infection With IFN-γ Macrophage Activation

Unlike opportunistic fungal pathogens, Histoplasma capsulatum can infect and cause disease in otherwise healthy individuals. Infections result from inhalation of infectious fungal conidia (spores) following disturbance of soils where it grows as a mycelium.

In the lung, exposure to the mammalian body temperature triggers the conidia to convert into pathogenic yeast cells which invade phagocytic cells of the immune system. Normally, these phagocytic cells are adept at eliminating fungal invaders, but H. capsulatum yeasts are able to survive and proliferate within these immune cells. With time, and only after generation of adaptive immunity, these phagocytic cells become activated, eventually leading to control of the pathogen. How H. capsulatum grows within phagocytic cells and how the macrophage changes during activation to control the infection are not well understood.

We identified a copper transporter (Ctr3) that contributes to H. capsulatum growth in macrophages. H. capsulatum can grow in high levels of copper, normally toxic to other microbes, but the Ctr3 transporter enables H. capsulatum to acquire copper when copper becomes limiting. During the innate immune response, macrophages are unable to control H. capsulatum and we found that Ctr3 was not required during this stage of infection. However, the onset of adaptive immunity and subsequent activation of macrophages led to rapid control of a Ctr3-deficient strain. These findings suggest that during early stages of the immune response, there is sufficient copper within the macrophage, but that activation of macrophages causes restriction of copper from H. capsulatum in the macrophage.

To verify this, we created a strain of H. capsulatum as a biosensor for copper levels in the H. capsulatum-containing compartment within the macrophage. We constructed a copper-sensing probe by fusing the copper-responsive CTR3 promoter with green fluorescence protein to estimate copper concentrations in H. capsulatum– infected macrophages. In resting and alveolar macrophages, which H. capsulatum first encounters, the H. capsulatum-containing compartment had high levels of copper, thereby permitting H. capsulatum growth. However, treatment of with pro-inflammatory IFN-γ created a copper-restricted environment for intracellular H. capsulatum cells. This change from a copper-high to a copper-limiting intracellular compartment was substantiated in vivo during lung infection.

These findings demonstrate that the macrophage initially infected by H. capsulatum during infection (i.e., during the innate immunity stage), has ample copper which supports intracellular fungal growth but is not toxic to H. capsulatum. With the later onset of adaptive immunity, the production of IFN-γ activates the macrophages that include switching to a copper-restricted intracellular environment in order to limit the growth of intracellular pathogens. Thus, macrophages use both high and low copper in the attempt to control pathogens by copper toxicity or nutritional immunity, respectively. We show for the first time that the activation of immune cells to control fungal pathogens mechanistically includes restriction of available copper, forcing intracellular H. capsulatum to rely on Ctr3 transporter to maintain fungal copper homeostasis.