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Phagosomal proteolysis in dendritic cells is modulated by NADPH oxidase in a pH-independent manner.

ABSTRACT: The level of proteolysis within phagosomes of dendritic cells (DCs) is thought to be tightly regulated, as it directly impacts the cell's efficiency to process antigen. Activity of the antimicrobial effector NADPH oxidase (NOX2) has been shown to reduce levels of proteolysis within phagosomes of both macrophages and DCs. However, the proposed mechanisms underlying these observations in these two myeloid cell lineages are dissimilar. Using real-time analysis of lumenal microenvironmental parameters within phagosomes in live bone marrow-derived DCs, we show that the levels of phagosomal proteolysis are diminished in the presence of NOX2 activity, but in contrast to previous reports, the acidification of the phagosome is largely unaffected. As found in macrophages, we show that NOX2 controls phagosomal proteolysis in DCs through redox modulation of local cysteine cathepsins. Aspartic cathepsins were unaffected by redox conditions, indicating that NOX2 skews the relative protease activities in these antigen processing compartments. The ability of DC phagosomes to reduce disulphides was also compromised by NOX2 activity, implicating this oxidase in the control of an additional antigen processing chemistry of DCs.

SUBMITTER: Rybicka JM

PROVIDER: S-EPMC3280544 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Phagosomal proteolysis in dendritic cells is modulated by NADPH oxidase in a pH-independent manner.

Rybicka Joanna M JM Balce Dale R DR Chaudhuri Sibapriya S Allan Euan R O ER Yates Robin M RM

The EMBO journal 20111213 4

The level of proteolysis within phagosomes of dendritic cells (DCs) is thought to be tightly regulated, as it directly impacts the cell's efficiency to process antigen. Activity of the antimicrobial effector NADPH oxidase (NOX2) has been shown to reduce levels of proteolysis within phagosomes of both macrophages and DCs. However, the proposed mechanisms underlying these observations in these two myeloid cell lineages are dissimilar. Using real-time analysis of lumenal microenvironmental paramete ...[more]

PMID: 22157818

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Project description:Neutrophils migrating from the blood (pH 7.35-7.45) into the surrounding tissues encounter changes in extracellular pH (pHe) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H+ ions reducing the intracellular pH (pHi). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pHe (ranging from 6.6 to 7.8; every 0.2 units) increased pHi of both activated and resting neutrophils within 10-20?min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H+ ions, pHi is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pHe promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pHe-mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H+ ions, whereas each bicarbonate HCO3- ion binds 1H+ ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar bicarbonate solution. For that reason, regulating NETosis by pH with specific buffers such as THAM could be more effective than bicarbonate in managing NET-related diseases.

Dataset Information

Phagosomal proteolysis in dendritic cells is modulated by NADPH oxidase in a pH-independent manner.

Publications

Phagosomal proteolysis in dendritic cells is modulated by NADPH oxidase in a pH-independent manner.

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