Flavocytochrome b 558 , the catalytic core of the phagocytic NADPH oxidase, mediates the transfer of electrons from NADPH to molecular oxygen to generate superoxide for host defense. Flavocytochrome b is a membrane heterodimer consisting of a large subunit gp91 phox (NOX2) and a smaller subunit, p22 phox . Although in neutrophils flavocytochrome b has been shown to localize to the plasma membrane and specific granules, little is known about its distribution in macrophages. Using immunofluorescent staining and live cell imaging of fluorescently tagged gp91 phox and p22 phox , we demonstrate in a Chinese hamster ovary cell model system and in RAW 264.7 and primary murine bone marrow-derived macrophages that flavocytochrome b is found in the Rab11-positive recycling endocytic compartment, as well as in Rab5-positive early endosomes and plasma membrane. Additionally, we show that unassembled p22 phox and gp91 phox subunits localize to the endoplasmic reticulum, which redistribute to the cell surface and endosomal compartments following heterodimer formation. These studies show for the first time that flavocytochrome b localizes to intracellular compartments in macrophages that recycle to the plasma membrane, which may act as a reservoir to deliver flavocytochrome b to the cell surface and phagosome membranes. T he generation of superoxide by the phagocytic NADPH oxidase provides antimicrobial defense essential to innate immunity. The NADPH oxidase is a multicomponent enzyme, comprised of a membrane-bound heterodimer, flavocytochrome b 558 , which consists of a large subunit, gp91 phox (NOX2), and a small subunit, p22 phox , and cytosolic regulatory subunits p67 phox , p47 phox , p40 phox , and Rac (1). Genetic deficiencies in the NADPH oxidase impair superoxide production and result in chronic granulomatous disease, which is characterized by a marked inability to kill certain microorganisms, resulting in frequent and life-threatening fungal and bacterial infections (2). Proper targeting of the NADPH oxidase complex is also required for microbe killing. Thus, while several pathogens prevent NADPH oxidase assembly at the phagosome in macrophages and/or neutrophils as a means to evade killing by toxic oxidants (3-7), others such as Helicobacter plyori (8) disrupt enzyme targeting such that active NADPH oxidase complexes accumulate at the cell surface, and superoxide is generated in the extracellular space instead of the phagosome lumen.The subcellular distribution of flavocytochrome b has been well characterized in neutrophils. gp91 phox and p22 phox are synthesized as separate polypeptides in the endoplasmic reticulum (ER). 3 Human gp91 phox is generated from a core protein of 58 kDa (9) that is subsequently glycosylated to gp65, a high-mannose 65-kDa form in the ER that binds to heme, allowing for heterodimer formation with p22 phox (10 -13). The heterodimer then traffics to the Golgi where gp91 phox is further glycosylated to the mature, 91-kDa form (13). In resting neutrophils, the mature flavocytochrome is found p...

Highlights d Combination checkpoint blockade leads to impaired efficacy with low tumor burden d This impairment results from IFN-g-mediated deletion of tumor-reactive T cells d AICD is an immune-intrinsic mechanism of therapeutic resistance to checkpoint blockade

IFNγ regulates trafficking and synthesis of flavocytochrome b558, suggesting a role to control superoxide production in macrophages. 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, gp91phox, and a smaller subunit, p22phox. We recently showed in murine macrophages that flavocytochrome b558 localizes to the PM and Rab11-positive recycling endosomes, whereas in primary hMDMs, gp91phox and p22phox 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 gp91phox and p22phox but also demonstrate that IFN-γ induced a shift in the predominant localization of gp91phox and p22phox 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.

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