Abstract
Current Concepts
It is universally accepted that the primary function of the generation of ROS and RNI products by phagocytes is to kill microorganisms. These products may be confined to the phagosome or released in the extracellular environment. No individual cell does this better than the neutrophil. In this Forum, Winterbourn and Kettle comprehensively review the particular oxidants produced within the phagosomes of neutrophils, and their proposed microbicidal actions (9). Although much is known about these chemistries in isolation, the complexities and dynamics of their function within the phagosomal lumenal microenvironment is less defined. Equally less defined is the multitude of signaling events that lead to NOX2 assembly and activity on these phagosomes. In addition, in this Forum, Bréchard et al. outline the currently-known signaling pathways responsible for NOX2 function on the neutrophil's phagosome after uptake via FcγR and CR3 (1). This review particularly focuses on the phospholipid- and Ca2+- dependent pathways and the potential roles of S100A8/S100A9 in these signaling cascades.
Over the past decade, it has become abundantly clear that the respiratory burst itself is not a terminal event, but is a key player in the orchestration of downstream biological processes, both intracellularly and extracellularly. Intracellularly, NOX2 activity has been implicated in an ever-growing number of signaling pathways within phagocytes. In the forum review by Vernon and Tang, the authors summarize the downstream consequences of NOX2 activation to trafficking events within the phagocyte (8). In particular, they discuss the relationship between the respiratory burst and autophagy. In contrast, Nussbaum et al. explore the extracellular consequences of the release of neutrophil-derived oxidants and redox-active enzymes such as myeloperoxidase (MPO), focusing on the role of MPO in modulating acute and chronic inflammation (5).
Current Controversies
It has been reported by a number of groups, and generally agreed on, that NOX2 activity negatively impacts proteolysis within the phagosome of macrophages and DCs, and that this has significant consequences on antigen processing within these compartments. However, the mechanism/s by which this occurs, specifically in DCs, is less black and white. As reviewed in this Forum, work done by the Amigorena research group supports a mechanism mediated through significant NOX2-mediated alkalinization of the DC phagosome (beyond the optimum of the majority of the lysosomal cathepsins) (4). While this is an attractive hypothesis, since NOX2 function and proton translocation are electrogenically coupled, the proposed mechanism is reliant on a yet-unidentified, non-proton compensating counter-ion flux that could allow NOX2 activity to alkalinize the lumen. Such large, osmotically active counter-ion fluxes are theoretically problematic in a closed system such as the phagosome (3). More recent work done within my laboratory found NOX2-mediated inhibition of phagosomal proteolysis without significant perturbation of phagosomal acidification in all three of the DC models studied (7). In this body of work, it was demonstrated that, as in macrophages, NOX2 activity impacted proteolytic rates within the phagosome through oxidative inactivation of local cysteine cathepsins. The discrepancies between these two studies have yet to be elucidated. Nevertheless, while the proposed mechanisms may be dissimilar, it is clear that both Amigorena's and my group agree that NOX2 modulation of phagosomal proteolysis in DCs is an important process which warrants further investigation and open discussion.
Future Challenges
Much of the past and current research efforts on redox control of the phagosome has focused on the phagosome's oxidative machinery. Conversely, there has been little productive work done to investigate the mechanisms that generate the reductive potential of phagosomes, endosomes, and lysosomes. Generally, it is accepted that the lumens of these vacuoles are predominantly reductive and that the reductive potential of these microenvironments is essential to their function (2, 6). However, there are little to no solid data which point toward what mechanisms or machinery create and maintain the reductive potential of these microenvironments. It has been suggested that the reductive potential of lysosomes is maintained by a cysteine transporter which produces a net influx of cysteine, paired with a transporter that effluxes cystine (oxidized cysteine) (6). However, since the proposed existence of this system in the early 1990s, the stoichiometry of such a mechanism has been disputed, and the lysosomal cysteine transporter (on which the proposed mechanism relies) has not been identified, suggesting the existence of an alternate source of the lysosome's reductive potential. Despite the undisputed importance of the reductive potential and of disulfide reduction within these compartments, the lack of convenient and accurate methods for measurement of this feature has resulted in its placement in the proverbial “too-hard basket.” As assays that permit real-time measurement of phagosomal and lysosomal disulfide reduction come to the fore, they will allow us to explore this frontier of phagosomal biology more effectively.