Effects of IFN-γ on intracellular trafficking and activity of macrophage NADPH oxidase flavocytochrome b₅₅₈

Flavocytochrome b₅₅₈, the catalytic core of the phagocyte NADPH oxidase (NOX2), mediates electron transfer from NADPH to molecular oxygen to generate superoxide, the precursor of highly ROS for host defense. Flavocytochrome b₅₅₈ is an integral membrane heterodimer consisting of a large glycosylated subunit, gp91^phox, and a smaller subunit, p22^phox. We recently showed in murine macrophages that flavocytochrome b₅₅₈ localizes to the PM and Rab11-positive recycling endosomes, whereas in primary hMDMs, gp91^phox and p22^phox reside in the PM and the ER. The antimicrobial activity of macrophages, including ROS production, is greatly enhanced by IFN-γ, but how this is achieved is incompletely understood. To further define the mechanisms by which IFN-γ enhances macrophage NADPH oxidase activity, we evaluated changes in flavocytochrome b₅₅₈ expression and localization, along with NADPH oxidase activity, in IFN-γ stimulated RAW 264.7 cells and primary murine BMDMs and hMDMs. We found that enhanced capacity for ROS production is, in part, a result of increased protein expression of gp91^phox and p22^phox but also demonstrate that IFN-γ induced a shift in the predominant localization of gp91^phox and p22^phox from intracellular membrane compartments to the PM. Our results are the first to show that a cytokine can change the distribution of macrophage flavocytochrome b₅₅₈ and provide a potential, new mechanism by which IFN-γ modulates macrophage antimicrobial activity. Altogether, our data suggest that the mechanisms by which IFN-γ regulates antimicrobial activity of macrophages are more complex than previously appreciated.