NADPH oxidase activity controls phagosomal proteolysis in macrophages through modulation of the lumenal redox environment of phagosomes

Abstract

The phagosomal lumen in macrophages is the site of numerous interacting chemistries that mediate microbial killing, macromolecular degradation, and antigen processing. Using a non-hypothesis-based screen to explore the interconnectivity of phagosomal functions, we found that NADPH oxidase (NOX2) negatively regulates levels of proteolysis within the maturing phagosome of macrophages. Unlike the NOX2 mechanism of proteolytic control reported in dendritic cells, this phenomenon in macrophages is independent of changes to lumenal pH and is also independent of hydrolase delivery to the phagosome. We found that NOX2 mediates the inhibition of phagosomal proteolysis in macrophages through reversible oxidative inactivation of local cysteine cathepsins. We also show that NOX2 activity significantly compromises the phagosome's ability to reduce disulfides. These findings indicate that NOX2 oxidatively inactivates cysteine cathepsins through sustained ablation of the reductive capacity of the phagosomal lumen. This constitutes a unique mechanism of spatiotemporal control of phagosomal chemistries through the modulation of the local redox environment. In addition, this work further implicates the microbicidal effector NOX2 as a global modulator of phagosomal physiologies, particularly of those pertinent to antigen processing.

Fig. 1.

Reactive oxygen species generation by phagosomal NOX2 decreases levels of phagosomal proteolysis in macrophages. Oxidative burst and bulk proteolytic activity within macrophage phagosomes were evaluated following phagocytosis of fluorescently labeled, IgG-coupled experimental particles. BMMØs were treated with 0.5 μM DPI, 10 μM THF, 25 μM quercetin (QUE), or DMSO alone for 1 h before phagocytic uptake. (A and B) Phagosomal oxidative burst was assessed by measurement of fluorescence liberated by oxidation of particle-associated H2HFF-OxyBURST substrate relative to calibration fluorescence in IFNγ-activated BMMØs. Phagosomal bulk proteolysis was assessed by measurement of fluorescence liberated through hydrolysis of particle-associated DQ-albumin relative to calibration fluorescence in resting (C and D) and IFNγ-activated (E and F) BMMØs. (A, C, and E) Real-time representative traces. Relative fluorescent units (RFU) are proportional to the degree of substrate oxidation/hydrolysis. (B, D, and F) Averaged rates/activities relative to DMSO-treated WT samples. Rates/activities were determined through calculation of the gradient of the linear portion of the real-time trace (as described by y = mx + c, where y = relative fluorescence, m = gradient, and x = time) relative to DMSO-treated WT samples. (D and F) Graphs represent averaged data from three independent experiments. Error bars denote SEM. P values were determined by one-way analysis of variance (ANOVA).

Fig. 2.

NOX2 activity does not affect phagosomal acidification, phagosome-lysosome communication, or phagosomal β-galactosidase activity in macrophages. (A and B) Phagosomal pH following phagocytosis was calculated using excitation ratio fluorometry of the pH-sensitive carboxyfluorescein on IgG-coupled beads followed by regression to a standard curve. (A) Representative acidification profiles in resting BMMØs. (B) Final lumenal pH at 30 min postinternalization in resting BMMØs from four independent experiments. Error bars represent SEM. (C) Profile of phagosome-lysosome communication in real time using FRET efficiency between a particle-restricted donor fluor and a fluid-phase lysosomal acceptor fluor. Relative fluorescent units (RFU) correlate to the concentration of lysosomal constituents within the phagosome at a given point in time. (D) Profile of β-galactoside hydrolysis in phagosomes in real time.

Fig. 3.

NOX2 activity negatively regulates cysteine but not aspartic cathepsin activity in the phagosome. Relative activities of phagosomal proteases were evaluated using cathepsin D/E- and B/L-specific fluorogenic peptides bound to IgG-coupled experimental particles in the presence or absence of NOX2 activity. Phagosomal cathepsin D/E (aspartic cathepsins) (A and B) and cathepsin B/L (cysteine cathepsins) (C and D) activities in resting BMMØs. (A and C) Real-time representative traces. (B and D) Averaged rates between 15 and 40 min postinternalization, relative to DMSO-treated WT samples, from three independent experiments. Error bars represent SEM. P values were determined by ANOVA.

Fig. 4.

NOX2 inactivates phagosomal cysteine cathepsins via a reversible oxidative modification. Aspartic and cysteine cathepsin activities of phagosomes isolated from WT and Cybb^−/− BMMØs ± 0.5 μM DPI were measured fluorometrically in vitro, with or without reduction by 1 μM DHLA and 30 mM GSH. (A–C) Relative activities were determined by the rate of increase in fluorescence of cathepsin-specific fluorogenic substrates at 37 °C, pH 5.5 and expressed relative to the corresponding Cybb^−/− samples. (A) Cathepsin B (cysteine cathepsin); (B) cathepsin S (cysteine cathepsin); (C) cathepsin D/E (aspartic cathepsins). Graphs represent data from three independent experiments. Error bars denote SEM. P values were determined by ANOVA. (D) Relative proportions of active cathepsin B in phagosomes isolated from WT and Cybb^−/− BMMØs were determined by reaction with the cathepsin B-specific biotinylated irreversible inhibitor biotin-FA-FMK with or without reduction by DHLA and GSH. Western blot images depict active (biotinylated) and total cathepsin B.

Fig. 5.

NOX2 activity diminishes the reductive capacity of the phagosome. Phagosomal reductive capacity was assessed by measurement of fluorescence liberated through reduction of a modified fluorogenic cystine-based reagent covalently bound to IgG-coupled experimental particles in resting (A and B) and IFNγ-activated (C and D) BMMØs. (A and C) Real-time representative traces. (B and D) Averaged rates relative to DMSO-treated WT samples from three independent experiments. Error bars denote SEM. P values were determined by ANOVA.