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

Flavocytochrome b(558), 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 b(558) 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 b(558) 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-gamma, but how this is achieved is incompletely understood. To further define the mechanisms by which IFN-gamma enhances macrophage NADPH oxidase activity, we evaluated changes in flavocytochrome b(558) expression and localization, along with NADPH oxidase activity, in IFN-gamma 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-gamma 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 b(558) and provide a potential, new mechanism by which IFN-gamma modulates macrophage antimicrobial activity. Altogether, our data suggest that the mechanisms by which IFN-gamma regulates antimicrobial activity of macrophages are more complex than previously appreciated. J. Leukoc. Biol. 92: 869-882; 2012.