Oxidized phagosomal NOX2 complex is replenished from lysosomes

Abstract

In dendritic cells, the NADPH oxidase 2 complex (NOX2) is recruited to the phagosomal membrane during antigen uptake. NOX2 produces reactive oxygen species (ROS) in the lumen of the phagosome that kill ingested pathogens, delay antigen breakdown and alter the peptide repertoire for presentation to T cells. How the integral membrane component of NOX2, cytochrome b558 (which comprises CYBB and CYBA), traffics to phagosomes is incompletely understood. In this study, we show in dendritic cells derived from human blood-isolated monocytes that cytochrome b558 is initially recruited to the phagosome from the plasma membrane during phagosome formation. Cytochrome b558 also traffics from a lysosomal pool to phagosomes and this is required to replenish oxidatively damaged NOX2. We identified syntaxin-7, SNAP23 and VAMP8 as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediating this process. Our data describe a key mechanism of how dendritic cells sustain ROS production after antigen uptake that is required to initiate T cell responses.

Endosomal and Phagosomal SNAREs

“…The Qa-SNARE Stx7 mainly locates at late endosomes and lysosomes (17,24,62,88,217), but is also found at early and recycling endosomes (29,88,158,247) and the plasma membrane (134,158). In addition, Stx7 locates at phagosomes, as found by imaging (62,79), Western blot (271), and proteomics (38,43,156,286,293). Although Stx7 can be recruited to phagosomes in multiple phases (267), several studies showed a rapid increase in phagosomal Stx7 over time after uptake (Refs.…”

“…The Stx7-Vti1b-Stx8 SNARE complex with VAMP7 (R) instead of VAMP8 mediates fusion of late endosomes with lysosomes and homotypic fusion of lysosomes (251,322). Stx7 also mediates fusion of lysosomes with phagosomes (24,79) and plays a role in trafficking from the trans-Golgi network to endosomes (57).…”

“…Superresolution-stimulated emission depletion microscopy revealed that Stx12 can organize in membrane domains at endosomal membranes (107). Stx12 also locates at phagosomes (38,43,62,79,96,156,271,286), and, although one study showed that Stx12 was rapidly cleared from phagosomes after uptake (62), most studies report no or only a slow clearance of Stx12 from phagosomes (Refs. 79, 96, 271 and FIGURE 5).…”

“…The Qb-and Qc-SNARE-motifs of SNAP23 are connected by a linker region containing five palmitoylated cysteines (272) that anchor it at the plasma membrane, recycling and late endosomes, and the trans-Golgi network (88,94,104,109,166,223,275,309,312,346). SNAP23 is also found at phagosomes (38,51,79,215,221,271,286), and, although it might be recruited at several stages during phagosomal maturation (267), most quantitative proteomics show an overall decrease of phagosomal SNAP23 following uptake (FIGURE 5). Although best understood for its role in plasma membrane trafficking, SNAP23 also catalyzes endosomal fusion events (166,309), such as for the biogenesis of lysosome-relate organelles (e.g., melanosomes) (339).…”

Independent trafficking of flavocytochrome b558 and myeloperoxidase to phagosomes during phagocytosis visualised by energy‐filtering and energy‐dispersive spectroscopy‐scanning transmission electron microscopy

“…Enzymes of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) family are important sources of reactive oxygen species (ROS) (Brandes et al ., ). Phagocytosis‐associated Nox plays a crucial role in host defenses involving neutrophils, monocytes, and macrophages (Dingjan et al ., ; Minakami & Sumimotoa, ). ROS production is mediated by Nox, which transfers electrons to the extracellular space in converting oxygen to O 2 − (Okochi Y et al ., ).…”