Reactive Oxygen Species Production in the Phagosome: Impact on Antigen Presentation in Dendritic...

FORUM REVIEW ARTICLE

Reactive Oxygen Species Production in the Phagosome:Impact on Antigen Presentation in Dendritic Cells

Fiorella Kotsias,* Eik Hoffmann,* Sebastian Amigorena, and Ariel Savina

Abstract

Significance: The NADPH oxidase 2 (NOX2) is known to play a major role in innate immunity for several decades.Phagocytic cells provide host defense by ingesting microbes and destroy them by different mechanisms, includingthe generation of reactive oxygen species (ROS) by NOX2, a process known as oxidative burst. The phagocyticpathway of dendritic cells (DCs), highly adapted to antigen processing, has been shown to display remarkabledifferences compared to other phagocytes. Contrary to macrophages and neutrophils, the main function of DCphagosomes is antigen presentation rather than pathogen killing or clearance of cell debris. Recent Advances: Inthe last few years, it became clear that NOX2 is also involved in the establishment of adaptive immunity. Severalstudies support the idea of a relationship between antigen presentation and the level of antigen degradation, thelatter one being regulated by the pH and ROS within phagosomes. Critical Issues: The regulation of phagosomalpH exerted by NOX2, and thereby of the efficacy of antigen cross-presentation in DCs, represents a clear illus-tration of how NOX2 can influence CD8+ T lymphocyte responses. In this review, we want to put emphasis on therelationship between ROS generation and antigen processing and presentation, since there is growing evidencethat the low levels of ROS generated by DCs play an important role in these processes. Future Directions: In thenext years, it will be interesting to unravel possible mechanisms involved and to find other possible connectionsbetween NOX family members and adaptive immune responses. Antioxid. Redox Signal. 18, 714–729.

Introduction

Phagocytosis is an active process by which microor-ganisms, dying cells, or cell debris are engulfed and

internalized by different types of cells, collectively calledphagocytes. This selective group of cells includes macro-phages, neutrophils, and dendritic cells (DCs). They scavengebody fluids, peripheral, and lymphoid tissues and constantlysample their environment. The process of phagocytosis rep-resents not only one of the earliest host responses of innateimmunity against pathogens but also a critical process for theinitiation and modulation of adaptive immune responses.Therefore, defense strategies of phagocytes are supported bydifferent mechanisms, including the production of reactiveoxygen species (ROS) by the NADPH oxidase (NOX) com-plex. The generation of ROS by neutrophils and macrophages,also known as respiratory burst, is a crucial event in pathogenkilling (8). On the other hand, DCs are not considered ascritical players of the first line of immune defense. Contrary tomacrophages and neutrophils, phagocytosis and phagosomalROS production by DCs serve other purposes than the mere

clearance of microbes or dead cells. DCs are highly specializedin antigen processing and presentation (112). Consequently,their main function is to initiate adaptive immune responsesthrough the activation of naıve T lymphocytes. T cell activa-tion is triggered when major histocompatibility complex(MHC) molecules, both class I (MHC-I) and class II (MHC-II),loaded with specific immunogenic peptides and exposed onthe DC surface, engage their cognate T cell receptor. Contraryto MHC-II/peptide complexes that are restricted to the acti-vation of CD4 + T cells, MHC-I/peptide complexes exclu-sively activate CD8 + T cells, triggering cytotoxic immuneresponses (30). MHC-II/peptide complexes are commonlygenerated intracellularly by processing of internalized anti-gens (58, 91). Nevertheless, the source of peptides for MHC-Iloading can either be endogenous, from cytosolic antigenssuch as viral antigens (classical MHC-I presentation), or ex-ogenous, from phagocytosed antigens, a process known ascross-presentation (21, 30, 104).

There is collective evidence that DCs are the main antigen-presenting cells (APCs) that can cross-present antigens in vitroand in vivo (64). High efficiency in cross-presentation is, at

Institut Curie, INSERM U932, Paris Cedex 05, France.*These authors contributed equally to this work.

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least in part, a consequence of fine-tuned regulation of antigenprocessing, in which ROS production seems to play a criticalrole (61, 113, 114). Given that DCs represent a heterogeneouscell population, it is still unclear however which DC subtypesare the most efficient cross-presenting cells, in particular,under specific inflammatory situations.

In mice, spleen DCs (also known as resident DCs) can bedivided into two main populations according to the expres-sion of CD8aa at their cell surface (141). Therefore, CD8 + DCswere shown to be more efficient than CD8 - DCs in terms ofcross-presentation at steady state (122), whereas both sub-types presented antigens efficiently in the MHC-II context.However, other DC subsets, such as migratory DCs andCD103 + DCs, have also been shown to cross-present effi-ciently (32, 35). In humans, the situation of DC subsets is ascomplex and heterogeneous as in mice. BDCA3 + DCs wererecently proposed to be the homolog of murine lymphoidorgan-resident CD8 + DCs (5, 63), and therefore to be spe-cialized in cross-presentation. Among the human skin DCs,Langerhans cells and CD1a + DCs have been suggested to bethe most efficient cross-presenting cells (121).

It is most likely that the capacity to cross-present antigensbetween different DC subtypes is the final result of their intrinsicadaptation of intracellular pathways to this function, to theirsurrounding environment, and to the quality of the antigen.

In this review, we want to emphasize how phagosomalROS generation has an impact on antigen processing andpresentation.

The NOX Family

The NOX family consists of membrane-bound enzymesthat catalyze the reduction of oxygen to superoxide usingNADPH as an electron donor. The central component of allmembers of this family is a heterodimer of the product of theX-chromosomal cybb gene, the glycoprotein gp91phox (alsoreferred to as NOX2), and the cyba gene, p22phox (76). Thesetwo membrane components of the inactive NOX form theflavocytochrome b558 and will be referred as such in this re-view. In addition to the membrane components, the cytosolicsubunits p40phox, p47phox, and p67phox, as well as RacGTPases (Rac1 or Rac2 depending on the cell type), regulatethe activity of the NOX. In humans, the entire NOX familyconsists of NOX2 and six homologs: NOX1, NOX3, NOX4,NOX5, DUOX1, and DUOX2 (101).

NOX1 and NOX2 are the closest homologs of the family.NOX1 was identified in the colon, uterus, and prostate tissues,

in epithelial barriers, as well as vascular smooth muscle cells.NOX1 has been implicated in signaling cascades of angio-tensin-II, tumor necrosis factor a (TNFa), platelet-derivedgrowth factor, and epidermal growth factor. For full activa-tion in a cell type- and stimulus-dependent manner, NOX1requires two additional proteins, the organizer NOXO1and the activator NOXA1 (p47phox and p67phox homologs,respectively) (99).

NOX3 has been described in fetal tissues, the inner ear,hepatocytes, and macrophages and requires only p22phox andNOXO1 to generate ROS (26). However, participation ofNOXA1 is able to enhance ROS production, whereas the in-volvement of Rac1 in NOX3 regulation is still controversial(136). More recent data have shown that the regulation is Rac-dependent at similar levels compared to NOXA1 (65, 129).

NOX4 is constitutively active and does not need the inter-action with cytosolic regulatory subunits (65). It has beenfound in the kidney, osteoclasts, aortic endothelial cells, car-diac, and lung fibroblasts, as well as fetal tissues (101).

NOX5 has been described in human testis, spleen, lymphnodes, and endothelial and smooth muscle cells, but seems to beabsent in rodent tissues. Although it is able to bind p22phox,complex formation is not required for ROS production. Therecruitment of NOX5 to the plasma membrane requires PI (4,5)P2 and is regulated in Ca2 + -dependent pathways without theinvolvement of p22phox, p47phox, p67phox, and Rac (69, 70).

The dual oxidases DUOX1 and DUOX2 possess an addi-tional transmembrane segment with peroxidase homologyand are able to generate hydrogen peroxide while their in-volvement in superoxide production is not clear (99). They arehighly expressed in thyroid tissues and also seem to be in-volved in innate immunity in the gastrointestinal tract (51).

The Phagocyte Oxidase NOX2

The best-characterized member of the NOX family isNOX2, which has been described not only in neutrophils,macrophages, DCs, and eosinophils but also in non-phagocytic cells such as fibroblasts, endothelial cells, andcardiomyocytes (75). In resting conditions, it is highly glyco-sylated and resides in intracellular secondary granules and, atlower amounts, in the plasma membrane, whereas activationleads to translocation to the plasma membrane and phago-somal membranes (99). During activation, the whole complexis assembled by the recruitment of the cytosolic componentsp40phox, p47phox, p67phox, and Rac toward the transmem-brane component, the flavocytochrome b558 (Fig. 1).

FIG. 1. Schematic represen-tation of the assembly of thephagocyte oxidase NADPHoxidase 2 (NOX2). (A) In rest-ing cells, gp91phox and p22phoxare located at the plasmamembrane (PM) or in intracel-lular vesicles, whereas theother subunits remain cyto-solic. (B) After an activationsignal, the flavocytochromeb558 is translocated to the PM atthe site of the forming phago-some, and the cytosolic sub-units are recruited to assemblethe active oxidase complex.

ROS AND ANTIGEN PRESENTATION IN DENDRITIC CELLS 715

Rac proteins are members of the family of small GTPase,cellular proteins that cycle between a GDP-bound inactive state(cytosolic) and a GTP-bound active state (membrane-associat-ed). They have many disparate regulatory functions inphagocytes and are also key components of the NOX2 complexthat control its assembly (reviewed in 16). Upon cell activation,GDP is exchanged by GTP by mechanisms involving the ac-tions of GDP/GTP-exchange factors (GEFs). Rac binds directlyto p67phox (37) and also to the membrane through its C-ter-minal domain, giving proper orientation to p67phox within theoxidase complex (77). Two Rac members, Rac1 and Rac2,participate in the NOX assembly in phagocytic cells, althoughtheir roles appear to be dependent on the cell type and the cellactivation status. Rac2 was identified as a key player in theregulation of NOX2 in human neutrophils (72), and soon after,Rac1 was described in macrophages (1). In neutrophils, NOX2activation is dependent on Rac2, whereas Rac1, even thoughexpressed at similar levels, was proven to be dispensable for thecomplex activation and ROS production (48). Conversely, inmacrophages, Rac1 is by far the predominant isoform, andstudies carried out with cells from Rac2-deficient mice showthat this isoform is necessary for NOX2 activation and ROSproduction under some circumstances depending on the acti-vation stimulus (143). Recent work by Robin Yates’ group hasshown that the control of phagosomal functions, such as pro-teolysis, exerted by NOX2 was dependent on the activationstimulus in murine macrophages (6, 109).

Previous reports had already shown that, independently ofthe cell type, Rac1 is preferentially located at the plasmamembrane, whereas Rac2 was present on endomembranes(87). This different localization was later explained by thedifferent lipid composition of the membranes that regulatesprotein charge and thus affinity to membranes, conferring aspecific localization (146). These findings may explain howthe two Rac isoforms influence the localization site for theassembly of NOX2 in different cell types. This could be thereason why, in neutrophils, where Rac2 is the isoform in-volved in the complex, NOX2 is active mainly in phagosomesto exert its microbicidal function through the production ofintraphagosomal ROS. In contrast, in macrophages, whereRac1 is the isoform involved in the complex, NOX activationis mainly driven to the plasma membrane. As a result, themain phagosomal strategy for pathogen killing in this celltype is the activation of their proteolytic activity through animportant acidification rather than the respiratory burst. In-terestingly, Allen and colleagues showed that infection ofneutrophils with Helicobacter pylori retargets the NOX com-plex assembly from the phagosome to the plasma membraneas a survival strategy. The consequent release of ROS extra-cellularly provokes major tissue damage (2). A followingstudy described that H. pylori is also able to specifically acti-vate Rac1 in epithelial cells (67). This observation suggeststhat a similar situation may occur in H. pylori-infected neu-trophils and may provide additional evidence that the acti-vation of a specific Rac isoform would account for thelocalization of the NOX complex assembly.

Localization of NOX2 and ROS Productionin Different Phagocytes

Neutrophils, macrophages, and DCs are all phagocytes thatplay different functions in the immune response. Neutrophils

and macrophages are involved in the local inflammatory re-sponse and clearance that take place upon phagocytosis ofpathogens or infected cells. Additionally, macrophages, al-though not efficient in activating naıve T cells, can activatememory T cells (105, 112). DCs are mainly responsible forprocessing and presenting derived antigenic peptides to naıveT lymphocytes, triggering the adaptive immune response.

In resting neutrophils, the flavocytochrome b558 is foundmainly in the membrane of secondary granules (3, 66),whereas a smaller amount is located in the membranes of asubpopulation of peroxidase-negative granules (71). Uponactivation, translocation of flavocytochrome b558 occurs whengranules fuse with the phagosomal membrane or the plasmamembrane (18, 28, 47). As a consequence, extensive phos-phorylation of p47phox accompanies this translocation. Theoxidase activity is initiated after the phagosome is formed andoccurs intracellularly, while superoxide cannot be detectedextracellularly (119). The NOX2 complex can be found atdifferent states of activation: resting, primed, active, and in-active (39). The resting, unassembled oxidase is present incirculating cells and becomes primed upon a first stimulus,making the cell more susceptible to activation. Primingstimuli are neutrophil adhesion, bacterial lipopolysaccharides(LPS), and proinflammatory cytokines. Phosphorylation ofthe p47phox subunit is believed to be the key step to reach theprimed state (40), which can be quickly activated by a secondsignal even at a suboptimal concentration. The active, assem-bled form is the one found in cells at inflammatory sites and canbe induced by the pathogen itself or pathogen-derived prod-ucts. Inactivation of the complex through dephosphorylation ofsome of the subunits is a consequence of the action of anti-inflammatory agents that limit inflammation (40).

Derived from circulating monocytes, macrophages are tis-sue-localized phagocytes that have specific features andfunctions according to their localization. Nonetheless, alltypes of macrophages express gp91phox and p22phox as wellas the cytosolic subunits p47phox, p40phox, and p67phox. Inmonocyte-derived macrophages (10) and murine peritonealmacrophages (137), gp91phox was detected at the plasmamembrane, but also in intracellular vesicular structures. Hu-man monocyte-derived macrophages were described to re-cruit gp91phox to their phagosomal membranes afterinternalization of latex beads (84). Since macrophages lackspecific granules present in neutrophils, the acquisition ofNOX2 membrane components differs from the process de-scribed above. Phagosomes mature into phagolysosomes bysequential fusion and fission events exchanging membranesand soluble materials with endocytic compartments (36, 139).Maturation of phagosomes is driven by small GTPases of theRab family. Early events are controlled by Rab5, which isreplaced afterward by Rab7, to regulate the fusion with lateendosomes and lysosomes (103, 140). Rab11 is involved inmembrane recycling during this process (79). This model ofphagosome maturation by sequential fusion with endo-somes/lysosomes is generally accepted in the macrophagemodels. Nevertheless, this classical picture has been ques-tioned by some studies showing a more complex dynamic ofthe phagosome maturation. Recently, the subcellular locali-zation of NOX2 membrane components in macrophages hasbeen described. Unassembled monomers localize to the en-doplasmic reticulum (ER), and heterodimer formation is aprerequisite for efficient trafficking to target membranes (22).

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Using murine macrophages, the authors show that the fla-vocytochrome b558 localizes to the plasma membrane and tointracellular compartments, which are positive for Rab11.These recycle to the plasma membrane and reach nascentphagosomes from there, suggesting that the endocytic re-cycling compartment could be a reservoir of NOX2 membranecomponents in the same way that specific granules arethought to be the NOX2 storage site in neutrophils (22).

The generation of ROS in DCs was proposed by Matsueet al. (85), but Elsen and coworkers were the first to charac-terize the expression of the NOX2 complex in mouse splenicDCs (41). The very low amount of generated ROS led them toterm this activity as cryptic, since the amount of O2

- and H2O2

secreted was < 3% of that of neutrophils. They detected ROSproduction intracellularly only after concomitant treatmentwith the NOX activator phorbol-12-myristate-13-acetate(PMA) and toll-like receptor (TLR) agonists. Interestingly, theprotein detected with the gp91phox antibody, which has a sizeof 90–100 kDa in human and bovine neutrophils, migratedwith an apparent molecular mass of 60 kDa in murine DCs.The correct identity was confirmed, and the lower molecularmass explained by a difference in glycosylation (13). By im-munoprecipitation and confocal microscopy, the authorsshowed the association of p40phox, p47phox, and p67phox inthe cytosol and the localization of gp91phox and p22phox atthe plasma membrane in resting DCs (113). A more detailedstudy of the subcellular distribution of NOX2 components inmurine DCs was done later. In resting DCs, gp91phox wasdetected by confocal and immunoelectron microscopy at themembrane of vesicles throughout the cell (113). Furthermore,we also found gp91phox in Rab27a-positive vesicles that werealso positive for the lysosomal marker LAMP1. These vesi-cles were recruited to and fused with phagosomes througha Rab27a-dependent mechanism (61). By using Rab27a-deficient DCs (isolated from ashen mice), it was shown thatacquisition of the membrane components of the NOX com-plex by phagosomes was strongly delayed in absence ofRab27a. Recruitment of the flavocytochrome b558 to phago-

somes was concomitant with the recruitment of p47phox to thephagosome membrane, suggesting a functional assembly ofthe complex. As a consequence and as shown in Figure 2, theactivation of the complex measured by the phagosomal re-cruitment of the cytosolic p47phox was significantly affectedin Rab27a-deficient DCs compared to wt DCs and totallyabsent in DCs from gp91 -/- mice (Fig. 2). Similar resultswere obtained by functional assays, where the actual oxida-tive capacity of the phagosomal environment was measuredby using FACS-based assays applying a ROS sensor bound tobeads (113). NOX activity and ROS production were alsodescribed in human DCs. We have shown that after antigeninternalization, NOX2 complex activation occurs at themembrane of antigen-containing compartments (84).

As explained in the Introduction, different DC subsets havebeen identified in mouse lymphoid organs according to theexpression of specific surface markers (141). Splenic DCs canbe divided into CD8 + and CD8 - subsets, which have dis-tinctive oxidative capacities caused by a different localizationand assembly of NOX2 (114). CD8 - spleen DCs produce moretotal ROS than CD8 + DCs, and both subsets from cybb - / -

mice fail to show any activity, confirming that NOX2 is re-sponsible for ROS production in both cases. Consistent withthis, CD8 - DCs express higher amounts of gp91phox. How-ever, only CD8 + DCs were able to produce ROS at the pha-gosomal level, whereas phagosomes from CD8 - DCs failed toproduce ROS even after stimulation with PMA. CD8 + DCsrecruit gp91phox and p47phox at the phagosomal membrane,whereas CD8 - DCs do not recruit p47phox; thus, the NOX2complex cannot assemble (Fig. 3). These differences can beexplained by the role of Rac GTPases in the localization of theNOX complex (as described above). Although both cell typesexpress the two isoforms, CD8 + DCs displayed higher levelsof Rac2, whereas the expression of Rac1 was similar. Afterphagocytosis of latex beads, Rac2 accumulated stronglyaround the phagosomes of CD8 + , but not CD8 - cells, andRac1 was detected at the plasma membrane, around phago-somes and in vesicles in both CD8 + and CD8 - DCs. The

FIG. 2. NOX2 is efficientlyassembled and active in den-dritic cell (DC) phagosomes.Upper panel: p47phox wasdetected by immunofluores-cence and confocal micros-copy in wild-type DCs,Rab27a-deficient, and gp91phox-deficient DCs after 60 min ofphagocytosis of 3-lm latexbeads. Lower panel: quantifi-cation of gray level intensitiesof the lines shown in the in-sets of the upper panel.


presence of Rac2 at the phagosomal membranes exclusively inCD8 + DCs may account for the localization of the active NOXcomplex at the membrane of these intracellular compartmentsin this DC subset. In agreement with these findings, CD8 +

DCs from Rac2-deficient mice (where only Rac1 is present)showed a defect in recruiting NOX2 at the phagosomalmembrane, and the complex was relocalized to the plasmamembrane (probably through a Rac1-dependent mechanism).These observations suggest that Rac2 drives the assembly ofthe NOX2 complex to phagosomes of CD8 + DCs, whereasRac1 directs its activation to the plasma membrane in CD8 -

DCs. Given that both Rac isoforms are present in the differentsubsets, the differential expression may not be the only ex-planation for the different localization of the complex in thedifferent cell types. It is most likely that the specific DC subsetexpression of Rac effectors, GEFs, and GTPase-activatingproteins may account for favoring the activation of one Racisoform or the other one. As in macrophages and neutrophils,these differences in the Rac-induced assembly of NOX2among DC subsets would also reflect and contribute to theirdifferent functions during immune responses, as we willdiscuss later in this review.

Role of NOX2 in Immunity

The role of ROS produced by the NOX2 oxidase and thoseproduced thereafter by other enzymes, such as myeloper-oxidase, in bacterial killing has been extensively studied in

neutrophils (reviewed in 75, 101). First evidence came fromstudies investigating immune function of patients sufferingfrom chronic granulomatous disease (CGD). This diseaseresults in chronic and repeating intervals of bacterial andfungal infections and has been characterized by the absenceof establishing effective immune responses (9, 19). Pre-viously, different studies have reported that CGD patientsdisplay mutations in genes encoding gp91phox, p22phox,p47phox, and p67phox (reviewed in 56). The recognition ofbacterial products on the cell surface triggers engulfmentand bacterial destruction through the generation of a respi-ratory burst, which is characterized by high oxygen con-sumption initiating a rapid increase of ROS inside thephagocytic vacuole. However, the direct effect of ROS inbacterial killing has been questioned (119). The modulationof the environment of the phagosome was proposed to be thekey function of ROS in microbe killing. The ion flux thatoccurs to compensate the charges produced by the electro-genic NOX2 complex causes an increase in the ionic strengthand together with the changes induced in pH, produce therelease of granule proteins and ensure their correct functionin microbe killing (102, 119). In addition, chemotaxis ofneutrophils toward the site of infection has been also pro-posed to depend on NOX2-induced ROS generation, sinceneutrophils silenced for NOX2 or isolated from CGD pa-tients have impaired migration (55, 74). The involvement ofROS in the expression of chemokine receptors could alsocontribute to its effect on chemotaxis (78).

FIG. 3. NOX2 activity isdifferentially regulated inspleen DC subsets. p47phoxwas detected by immunoflu-orescence after 1 h of phago-cytosis of 3-lm latex beads inpurified CD8 + and CD8 -

DCs by confocal microscopy.The scale bar represents 5 lm.

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Although ROS and the oxidative burst have been linked tothe promotion of inflammation, increasing evidence pointstoward the idea that ROS act as a modulator of immune re-sponses (reviewed in 111). Data showing that individualswith CGD are more prone to autoimmune diseases and thatdefective ROS production in neutrophils is linked to an in-creased inflammatory response (46) support the regulatoryfunction of ROS in immunity using different autoimmunedisease models, such as arthritis and multiple sclerosis (94).

ROS production by NOXs has been linked to several sig-naling mechanisms that have immunological consequencesand has been extensively reviewed (44, 92). Signaling func-tions have been proposed to be the main role of the oxidativeburst in macrophages, where most of the superoxide pro-duced at the plasma membrane is dismutated outside the cells(45). A portion of the resulting peroxide could enter cellssurrounding the site to exert immunological functions. Al-though the permeability of membranes to peroxide is limited,there are other mechanisms through which peroxide couldenter cells in the small amounts that are necessary for sig-naling purposes (reviewed by 8, 11, 45, 111). Moreover, in-tracellular ROS have been shown to modify key factors thatcontrol the inducible expression of genes whose products arepart of the inflammatory response (50, 131).

In conclusion, the killing of intraphagosomal bacteriawithin phagosomes, which is the main function of neutrophilsand a key component of innate immunity, is dependent on theoxidative burst to provide the adequate environment forprotease function. This is in agreement with the fact that mostneutrophil NOX2 is present in granules of resting cells thataccess phagosomes soon after the internalization of microor-ganisms. The phagosomes of macrophages have a lower pH,which is considered necessary to exert its degradative func-tions. While their oxidative burst is lower than in otherphagocytes, the control exerted by phagosomal ROS in anti-gen proteolysis has been studied in detail (107, 109, 145).Considering that NOX2 assembles also at the plasma mem-brane in macrophages, it is not surprising that ROS functionas intra- and extracellular signaling molecules in macro-phages. In contrast, the main function of DCs is to process andpresent antigens and initiate and shape adaptive immuneresponses (112). In this sense, it is reasonable to expect thatNOX2 oxidase localization and production of ROS are regu-lated in a manner to serve this purpose. In the next sections,we will focus on the roles of NOX2 activity in antigen pro-cessing and presentation in DCs.

Role of Phagosomal ROS Production in AntigenPresentation in DCs

The phagocytic pathway of DCs, highly adapted to antigenprocessing, has been shown to display remarkable differencescompared to other phagocytes. Contrary to macrophages andneutrophils, the main function of DC phagosomes is antigenpresentation rather than pathogen killing or clearance of celldebris. Accordingly, their phagosomes are the source ofpeptides derived from extracellular antigens that will beprocessed and presented to T lymphocytes. As we mentionedearlier, DCs have the ability to present exogenous antigens toboth CD8 + and CD4 + T cells. Activation of CD8 + T cells viacross-presentation leads to cytotoxic immune responsesagainst a wide variety of exogenous antigens (including

tumor cells, viruses, and bacteria). Similarly, cross-presentationis believed to play an important role in maintaining theequilibrium between tolerance and immunity through thepresentation of self-antigens.

As explained in the Introduction, this pathway is almostexclusive to DCs, and the intracellular mechanisms are stillnot clear. After antigen uptake and probably a gentle earlydegradation, most proteins or polypeptides are transferred tothe cytosol, where they are processed by the proteasome andthen transported either back to the phagosome or to the ER tobe associated to MHC class I molecules (reviewed in 4, 30).These MHC class I/peptide complexes are then finallytransported and exposed at the cell surface to activate CD8 +

T cells. In contrast to this so-called cytosolic pathway of an-tigen cross-presentation, a vacuolar pathway has also beendescribed in which the antigen processing and loading ontoMHC class I occur without any cytosolic steps, within thephagosome (25).

On the other hand, MHC class II-restricted antigen pre-sentation is the process by which APCs activate specific CD4 +

T cells. In this pathway, the phagocytosed antigen is degradedwithin the phagosome that matures and fuses with lysosomesthat provide the necessary proteolytic enzymes. At the sametime, MHC class II molecules assembled in the ER are targetedto the endosomal–lysosomal pathway by the invariant chain(Ii), a chaperone that is associated to the MHC class II dimer,which prevents premature peptide loading. Once MHC classII molecules reach phagolysosomes, Ii is cleaved, and peptidesare loaded, and the MHC–peptide complex is transported tothe plasma membrane (reviewed in 91). Interestingly, recentresults also suggest a role of Ii in the regulation of cross-pre-sentation itself (7). Therefore, antigen processing is of centralimportance in DCs. The loading of antigenic peptides on bothMHC class I and II molecules requires tightly controlledproteolysis to preserve antigens from degradation. Severalmechanisms have been identified to explain the limitedphagocytic proteolysis in DCs, and a link between proteolysis,antigen processing, and antigen presentation has been pro-posed.

Early evidence of the regulation of the proteolytic activityamong different phagocytes came from the work of Lennon-Dumenil and coworkers, which assessed the protease activityof phagosomes in DCs and macrophages. They described thatphagolysosomal fusion is selectively regulated among thesedifferent phagocytes, observing that in DCs, there is lowerexpression and recruitment of proteases, and that the kineticsof phagosome maturation are faster in macrophages as com-pared to DCs (80). Later, a study of lysosomal functions de-scribed that the inefficient lysosome acidification in immatureDCs was due to limited recruitment of the V-ATPase to ly-sosomes, as compared to macrophages or mature DCs (135).Lower amounts of lysosomal proteases were also detected inDCs in vivo within mouse secondary lymphoid organs, as wellas in bone marrow-derived DCs (34). Lysosomal proteolyticactivity was decreased in murine DCs, as compared to mac-rophages, and lower proteolysis was the cause for enhancedantigen presentation. Further work showed that immunoge-nicity was enhanced by limiting the susceptibility to lyso-somal proteolysis in vivo (33). These results suggested thatantigen persists in a less-degradative phagosomal environ-ment in DCs, which would favor their efficient presentationon MHC class I and class II molecules.


Further evidence of this specialization of endocytic andphagocytic compartments in DCs came from the observationthat ROS production inside DC phagosomes regulates pha-gosomal properties. We showed that DC phagosomes con-taining latex beads actively alkalinize their lumen at pHvalues higher than 7 during the first hours after internaliza-tion (pH 7.5 after 60–120 min; 113). A clear alkalinization ofphagosomes reaching values of pH above 7.0 was also regis-tered using a DC cell line, DC2.4 (133). As originally proposedin neutrophils, ROS generation in DC phagosomes consumesan important amount of protons that have been provided bythe V-ATPase. This H + consumption provokes an increase inthe intraphagosomal pH. Indeed, compared to primarymacrophages and macrophage-like cell lines, DC phagosomeshad higher pH values, and this increase was dependenton NOX2 activity, because gp91phox-deficient DCs hadmore acidic phagosomes. In fact, p47phox assembly at themembrane was more sustained over time in DCs than inmacrophages. As a functional consequence of this particularphagosomal pH regulation, we showed that antigen cross-presentation of two different model antigens was decreased inDCs isolated from gp91phox-deficient mice. This was due toan increase in antigen degradation as a result of the altered pHvalues that induced higher activities of proteases that other-wise would be less active (113) (Fig. 4). The above-mentionedstudies used polystyrene beads as phagocytic cargo, butsimilar observations were made in murine bone marrow-derived DCs and macrophages containing Staphylococcusaureus. While in macrophages containing S. aureus only lowROS levels were generated, and their phagosomes acidifiedvery rapidly, the situation in DCs was different. DC phago-somes containing S. aureus exhibited much slower acidifica-tion profiles over time, whereas significantly more ROS weregenerated (Anna Sokolovska and Lynda M. Stuart, personalcommunication).

As mentioned above, the recruitment of vesicles contain-ing NOX2 membrane components to phagosomes wasdependent on Rab27a. Consequently, DCs from Rab27a-deficient ashen mice displayed a defect in antigen cross-presentation due to increased antigen degradation withinmore acidic phagosomes (61) (Fig. 5). Similar results werethen described for human DCs. Monocyte-derived DCs fromCGD patients displayed an internalization pathway withhigher acidification, increased proteolytic activity, and im-paired cross-presentation of antigens, as compared to DCsfrom healthy donors (84).

These studies supported the idea of a relationship betweenantigen presentation and the level of antigen degradation, thelatter one being regulated by the pH and ROS within pha-gosomes. Further evidence for the involvement of NOX2 andROS production on antigen presentation came from work inVav-deficient DCs. Vav is a member of the GEF family thatcatalyzes the exchange of bound GDP to GTP on Rac proteins(29). After stimulation through a variety of immune receptors,Vav undergoes tyrosine phosphorylation that is required forits exchange activity on Rac (17). Their role in ROS productionand the oxidative burst in macrophages had already beenestablished (88, 89). Vav-null DCs exhibited defects in ROSproduction comparable to gp91phox-deficient DCs, but incontrast responded to stimulation by PMA, suggesting thatNOX2 oxidase was functional (49). Analysis of phagosomalproperties in these cells showed a decrease in pH values and

FIG. 4. Changes in phagosomal and endosomal pH inbone marrow-derived dendritic cells (BMDCs) and mac-rophages (BMMØ). (A) Schematic illustration of changes inphagosomal pH after phagocytic uptake in wild-type cellscompared to gp91phox-deficient cells and its impact on cross-presentation (XP). (B) Differences in endosomal pH mea-sured by flow cytometry using a sensor fluorophore (as de-scribed in 115) after 10 min and 60 min of endocytosiscomparing wild-type and gp91phox-deficient BMDCs.Fluorescence of the pH sensor is decreasing during acidifi-cation of organelles.

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an increase in antigen degradation. DCs failed to cross-pres-ent the antigen bound to latex beads efficiently (49), thusproviding additional evidence of the link between NOX2function and antigen cross-presentation.

In line with these findings, further research was carried outwith different subsets of murine splenic DCs, which arecharacterized by different antigen presentation abilities; forexample, CD8 + DCs are known to cross-present more effi-ciently compared to CD8 - . This was shown to be partiallydue to the array of phagocytic receptors expressed (60, 118)and differences in their antigen-processing capacities (38,117). Our group found that the pH was higher in CD8 +

phagosomes compared to the ones of CD8 - DCs. Conse-quently, the proteolytic activity in CD8 - DCs was moredominant than in the CD8 + population. As described above,the differences observed in the regulation of phagoendosomalpH correlate with a different localization of the active NOX2complex. High phagosomal pH in CD8 + DCs correlates witha specific assembly of NOX on phagosomal membranes,whereas an acidic pH in CD8 - phagosomes corresponds tothe absence of the NOX assembly in these compartments (Fig.6) (114). As mentioned above, Vav1 regulates ROS productionand cross-presentation in DCs (49). Vav1 was previouslyshown to associate with p67phox and Rac2, but not Rac 1, inhuman neutrophils (89). The absence of Rac2 in phagosomesfrom CD8 - DCs could provide an explanation for the differ-ences observed between DC subsets according to the differ-ential expression of Rac isoforms. Surprisingly, CD8 - DCsproduce high levels of ROS at the plasma membrane thatcould serve other immune-related functions, such as extra-cellular signaling, chemotaxis, or modulation of the inflam-matory environment.

Interestingly, our studies in DCs showed that the regula-tion of intralumenal pH for efficient antigen processing is notrestricted to the phagocytic pathway. The endocytic pathwayin DCs is also highly adapted to antigen processing, and amechanism similar to that described for phagosomes proba-bly accounts for the regulation of the proteolytic activity inendosomes. We have observed that the pH in endosomes ismaintained around 7 and acidifies very slowly compared tothose from gp91phox-deficient DCs (114, Fig. 4B). Indeed, thecross-presentation of soluble proteins is also impaired in DCs

lacking gp91phox or Rab27a (61, 113). This was also confirmedin human DCs that displayed high co-localization of dextran(internalized by endocytosis) and p47phox, suggesting as-sembly and activation of the complex in endosomes (84).

Very recently, the group of R. Yates suggested a redox-based control instead of a pH-based regulation of phagosomal

FIG. 5. Phagosomes ofRab27a-deficient DCs dis-play more rapid kinetics ofphagolysosomal fusion com-pared to wild-type cells il-lustrated by the similarlocalization, but different in-tensity, of Lysotracker withphagosomes containing fluo-rescent latex beads. Fluores-cence microscopy imageswere acquired with identicalexposure times. The scale barrepresents 10 lm.

FIG. 6. Schematic illustration of the differential recruit-ment of the activated NOX2 complex in CD81 and CD82

DC subsets. While in CD8 + DCs (left), Rac2 directs assemblyof NOX2 to the phagosome, the activated complex cannot befound on phagosomal membranes of CD8 - DCs (right). In-stead, high levels of Rac1 induce an assembly at the PM ofCD8 - DCs. Therefore, phagosomes of CD8 - DCs are moreacidic than CD8 + phagosomes, which has essential influenceon their antigen presentation capabilities.


degradation in DCs (108). These authors observed strongphagosome acidification in mouse bone marrow-derived DCs(pH 5.5 after 10 min of internalization). These results resembletheir previous data obtained in macrophages (109). The dis-crepancy between the results of Yates and coworkers and theresult from others (49, 81, 133) and us (84, 113, 114) could bedue to multiple technical differences in the protocols used forthe generation of DCs and the measurement of the pH.Strikingly, Yates et al. used opsonized particles (IgG-coatedbeads) that undoubtedly trigger distinctive intracellularevents that may impact differently on the phagosomal pHregulation. Indeed, these authors also do not find any differ-ence in phagosome acidification between NOX2-defectiveand control DCs using IgG-coated beads, which is not unex-pected since IgG-containing phagosomes acidified stronglyeven in wild-type cells (which is not the case for phagosomescontaining non-opsonized particles). FccR engagement is wellknown to enhance phagosomal acidification and fusion tolysosomes (62, 132, 134). In any case, it is noteworthy that intheir assays for dynamic pH measurements (108, 144), theauthors employed techniques not usually associated with DCmanipulation, but with macrophages (DCs usually adherevery poorly to glass and do not resist washes).

In conclusion, there are a number of reports that point to-ward a regulation of antigen presentation by the phagosomalenvironment, specifically due to the effect of NOX2-generatedROS on phagosomal pH (49, 81, 84, 113, 114, 133). This isparticularly clear in the case of antigen cross-presentation. Therole of NOX2 and ROS in MHC class II-restricted antigenpresentation by DCs has not been analyzed in detail so far.Ebselen, a selenium-containing antioxidant compound thatinhibits NOXs, reduced the presentation of ovalbumin (OVA)to CD4 + T cells by DCs (85). Maemura et al. used Kupffer cells,antigen-presenting macrophages that reside in the hepaticsinusoids, to assess the role of ROS in antigen presentation.They found that OVA presentation to antigen-specific CD4 + Tcells was inhibited when blocking ROS production with di-phenylene iodonium (DPI), an inhibitor of NOXs, althoughthe role of ROS on antigen degradation and the phagosomalenvironment in this process was not investigated. They con-cluded that the effect of ROS on antigen presentation wasmainly due to its role as a second messenger (83). The workdone by our group has shown that presentation of OVA andHY antigens to CD4 + T cells was also defective in gp91phox-deficient DCs and, to a lesser extent, in Rac2-deficient DCs (114). In addition, Vav-null DCs were alsoimpaired in the presentation of bead-bound OVA to antigen-specific CD4 + T cells (49).

There is still a controversy about the compartments dedi-cated to MHC class I presentation. It is not clear whether theyare different from those dedicated to MHC class II-restrictedpresentation, or if they represent two different maturationstates of the same compartment (20, 120). In any case, therequirements for pH, ROS, and protease activities seem to bedifferent. The results showing that ROS production is re-quired for MHC class II presentation did not assess the role ofROS in antigen processing. Since ROS is also necessary forcross-presentation, it would be possible to speculate that ROSserve different functions in these two pathways, neutralizingthe pH to avoid excessive proteolysis in the case of cross-presentation and regulating other aspects of the MHC class IIpresentation pathway (Fig. 7).

Effects of ROS on Antigen PresentationIndependent of the pH

Until now, the study of the role of ROS production byNOX2 in antigen presentation by DCs has been focused on itsimpact on phagosomal pH and consequently on antigen fate.Most of these data support a regulatory function for ROSproduction to moderate the proteolytic environment by neu-tralizing the pH, thereby favoring antigen cross-presentation.

The fact that DCs assemble and activate NOX2 and pro-duce ROS constitutively at low levels suggests that ROS couldhave various biological functions in DCs. Besides the regula-tion of the pH in the internalization pathway, ROS may alsomodify, through direct oxidation, the molecular structure ofthe antigen, as well as proteins implicated in phagosomalfunctions. ROS are a group of highly reactive moleculeswith superoxide being the main component produced by the

FIG. 7. DC phagosomes serve as platform for antigenprocessing and presentation after phagocytic uptake ofexogenous, particulate antigens, such as microorganismsand infected cells. Processed antigenic peptides are loadedon either major histocompatibility complex (MHC) class I orII molecules, either directly or after export to the cytosol, andtranslocated to the cell surface to be presented to CD8 + andCD4 + T lymphocytes, respectively. These pathways of cross-presentation and MHC II presentation rely on the degrada-tive environment of the phagosome, which is influenced bythe proton influx of the V-ATPase, proton channels, anddifferent proteases. Reactive oxygen species (ROS) genera-tion by NOX2 regulates intraphagosomal pH to preserveantigens for efficient presentation. Since different toll-likereceptors (TLRs), such as TLR 2 and 4, are present at the cellsurface, they can be internalized together with the phago-cytic cargo or recruited specifically from intracellular com-partments. Therefore, their engagement and phagosomallocalization could modify NOX2 activity to couple ROSproduction and pH regulation to antigen processing of in-gested pathogens.

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NOX2 oxidase. Superoxide is very rapidly converted intohydrogen peroxide, either spontaneously or by the enzymaticaction of the superoxide dismutase, which can be found inphagosomes. Moreover, H2O2 also interacts with superoxidedirectly to produce singlet oxygen (52). Proteins containingcysteine residues are susceptible to oxidation by H2O2, sincethe thiol group at the side chain of cysteine can be oxidized toform a disulfide bond. If these cysteine groups are withinactive sites of enzymes, oxidation can change conformationand function of certain enzymes in a reversible fashion (12,110). In this sense, it has been shown previously that theV-ATPase could be oxidized to form a disulfide bond betweentwo cysteine residues within the nucleotide-binding subunitsof the enzyme, resulting in the reversible inactivation of thecomplex (42, 43). Likewise, singlet oxygen can inhibit serineand cysteine proteases (cathepsins B, L, and S, as well asdifferent caspases), but not aspartate proteases (cathepsins Dand E) and metalloproteases (90, 130). The proposed mecha-nism includes the action of singlet oxygen modifying an es-sential residue in the catalytic center that is common tocysteine proteases and prone to oxidative changes. Cysteineproteases play a key role in antigen degradation and aresubject to a complex regulation within the phagosome (57).

In addition to these possibilities, ROS could also have aregulatory impact on antigen presentation by direct oxidationof the antigen. This may induce molecular changes in itsstructure that might have an impact on the susceptibility toproteolytic degradation. One of the examples of regulationthrough oxidation of substrates is the production of the oxi-dized form of low-density lipoprotein by Toll-like Receptor(TLR)4-activated NOX1 in macrophages, a process that ac-celerates their conversion into foam cells, resulting in plaqueformation and atherosclerosis (23, 96). Cancer progression isanother example of how ROS can regulate proteolysis. Inmany cancer types, altered ROS production has an impact onprotease function and substrate susceptibility to their activity,which leads to an increase in extracellular matrix degradationand tumor invasion (reviewed in 125). In a recent study,presentation of OVA to T cells by DCs was enhanced by hy-pochlorous acid, a byproduct of ROS and myeloperoxidase,found in primary azurophil granules of neutrophils. Chlor-ination of OVA promoted its binding to cell membranes, thusaugmenting antigen uptake, as well as increased susceptibil-ity to proteolysis due to conformational changes in the proteinstructure (100). Modifications of antigen conformation due toROS and other byproducts could therefore influence proteinsusceptibility to proteolysis, affecting their transport to thecytosol or other steps of antigen processing. In line with this,the group of P. Cresswell has shown that the gamma-interferon (IFN)-inducible lysosomal thiol reductase (GILT),an antioxidant enzyme present in the endocytic pathway,favors presentation of antigens containing disulfide bondsthat upon reduction are processed to expose epitopes forMHC class II binding and are transported more efficiently tothe cytosol for cross-presentation (54;124).

Additionally, to the direct effect of ROS on proteins due totheir reactive nature, there are different indirect pathways bywhich ROS can regulate cell functions (reviewed in 73). Asmentioned before, ROS can inhibit phosphatases through re-dox-sensitive cysteine residues, and tyrosine phosphatasescontrol the phosphorylation state of numerous signal-trans-ducing proteins (128). The group of tyrosine phosphatases is

inactivated by H2O2, and this results in the activation ofseveral protein kinase pathways, which are normally in-hibited by tyrosine phosphatases. These are mechanismswhere ROS act as second messengers in signaling pathways,since they do not have the chemical structure to allow classicalreceptor ligand recognition.

Autophagosomes are a source of intracellular antigens forMHC class II-restricted presentation (95). Autophagy plays animportant role in antigen presentation (reviewed in 31). In astarvation-induced autophagy model, superoxide and H2O2

lead to autophagy by inhibiting the Atg4 cysteine proteaseoxidizing reversibly the cysteine residue within the enzymaticactive site (116). Atg4 is involved in the regulation of LC3lipidation, a post-translational modification critical for au-tophagy. Even though the source of ROS during starvation-induced autophagy is not from the phagosome, but mostlythe mitochondria, it provides another example of how ROScan modulate diverse cellular events through chemical mod-ifications of enzymes. Recent work published by Huang et al.showed that in the absence of NOX2 or after treatment of cellswith DPI, LC3 recruitment to the phagosome is impaired aftermicrobe-induced autophagy by bone marrow-derived neu-trophils (59). The authors hypothesized that ROS are medi-ating the assembly of the Atg5-Atg12-Atg16 complex, whichallows the localization of LC3 to the phagosomal membrane.

It is also tempting to speculate that ROS can influence otherevents of the intracellular membrane-trafficking machinerythat have been shown to be critical for antigen processing,notably for cross-presentation. Very recently, our group hasshown that ER proteins are recruited to the endophagocyticpathway through the intermediate compartment (ERGIC),allowing the molecular machinery of MHC class I-restrictedpresentation to access the phagosome (24). This recruitmentalso delays the fusion to lysosomes, favoring the controlleddegradation of antigens and resulting in efficient cross-pre-sentation. It would be very interesting to investigate whetherthe recruitment of ER proteins to DC phagosomes is somehowinfluenced by ROS, which diffuse or leak out of phagosomesand could act on cytoplasmic proteins that could function aseffectors or activators of Rab proteins or SNAREs that con-stitute the essential molecular machinery for intracellularmembrane fusion (reviewed in 127).

TLR Signaling and ROS Production in DCs

In addition to sensing and clearing pathogens, the innateimmune system has developed different strategies for pre-sentation of antigenic peptides to induce adaptive immuneresponses. TLRs are a type of pattern recognition receptors(PRRs) that identify pathogen-associated molecular patterns(PAMPs). Innate immunity and the functions of NOXs areclosely coupled. Important evidence came from observationsin CGD patients lacking the establishment of effective im-mune responses against invading microorganisms (reviewedin 93). The impact of TLR signaling and proinflammatorypathways on the activation of NOXs has been reported (86,98), suggesting a critical role of NOX isoforms and ROS pro-duction.

Cell activation can prime the NOX2 complex, which is ac-companied by protein phosphorylation and translocation.This complex assembly initiates a weak oxidative response,but the priming stimulus is not sufficient to induce the


oxidative burst itself (111). Priming stimuli include TLR ag-onists (LPS and flagellin), proinflammatory cytokines (TNF-aand Il-1b), and proteases (39). The relationship between hostimmunity, TLR signaling, and ROS generation has also beenfound for other members of the NOX family. Kawahara et al.have shown that NOX1 induced ROS production in a TLR4-dependent manner during H. pylori infection (67). Further-more, detection of Salmonella enteritides flagellin by TLR5 alsoexhibited higher ROS generation by NOX1 in intestinal epi-thelial cells (68). NOX3 is very abundant in lung tissues, andone report has shown that deficiency in TLR4 induced upre-gulation of NOX3, resulting in increased ROS generation andpulmonary emphysema (147). NOX4 is an important effectordownstream of TLR4 signaling, which includes direct inter-action between the two molecules and NFjB-dependentproinflammatory responses upon LPS engagement (97, 98).Additionally, this oxidase is implicated in the Toll/IL-1Rdomain-containing adaptor-inducing IFN-ß (TRIF)-mediatedactivation of the transcription factor IRF-3 (27), which is es-sential in the induction of interferons and IFN-stimulatedgenes.

As mentioned above, several pieces of evidence support arelationship between TLR signaling and different NOX familymembers in innate immunity. Microbial molecules triggerTLRs, favoring ROS production that results in pathogen kill-ing and inflammation. However, the selective regulation andsubcellular localization of NOXs in phagocytes pinpoint to anessential link between innate ROS generation and adaptiveimmune responses and put DCs in the focus. The work ofothers and us have shown that these cells control the phago-somal environment toward one of their main functions, thepresentation of antigenic peptides on MHC I and MHC IImolecules (112). Could TLR engagement in DCs contribute toa particular NOX2 regulation in phagosomes with differentconsequences in antigen processing through pH modulation(Fig. 7)? Little is known about the impact of TLRs on ROSgeneration in DCs, but different scenarios can be hypothe-sized. TLR stimulation causes DC maturation, which includeslong-lasting changes in their efficacy to process and presentantigens. Trombetta and colleagues have shown that theV-ATPase was more efficiently assembled on lysosomes, andits activity was enhanced after stimulation with LPS (33, 34,135). These results suggest that TLR4 stimulation has an im-pact on phagosome functions during DC maturation. Themodulation of phagosomal functions at early time points byTLR4 stimulation has been a matter of debate (15, 106), andthe eventual role of NOX2 in this system has not been eval-uated. Compared to neutrophils, DCs display lower NOXactivity (41). However, it has been shown that NOX activity inDCs is increased either by the interaction with TLR ligands(142) or by the interaction with antigen-specific T lympho-cytes (85). It is therefore likely that TLR engagement uponphagocytic uptake of microorganisms as well as phagosomalNOX activity need to be tightly controlled to allow effectiveantigen presentation.

The existence of different pathogens that inhibit NOX ac-tivity preventing ROS production could be used to gain in-sight into the relationship between TLRs and NOXs in termsof antigen presentation. Some pathogens such as Legionellapneumophila, Coxiella burnetii, and Yersinia pseudotuberculosisare able to prevent the NOX complex assembly by inhibitingaccumulation of its cytosolic components (53, 123, 126). Oth-

ers, such as Salmonella enterica (138), cause a mislocalization ofthe NOX, whereas Leishmania donovani abrogates the recruit-ment of specific cytosolic components of the NOX complex(82). It is not clear and will undoubtedly require exhaustivestudy whether pathogens can influence cross-presentationthrough the manipulation of NOX2 activity in DCs.

Differentiation of self from non-self is of essential impor-tance to mount immune responses. An interesting concept inthis field adds more complexity to this general picture. Thehypothesis of phagosomal autonomy proposes the autono-mous control of antigen processing and presentation withinmicrobe-containing phagosomes, but not in other phago-somes within the same cell (14). The involvement of TLRs inphagosomal autonomy and the detection of microbial non-self-, but not self-antigens, by TLRs remain a matter of debate(15, 106). However, it becomes evident that innate sensors domodulate the kinetics and efficacy of phagosome maturationand therefore antigen processing and loading in a phago-some-autonomous manner. This would represent a finediscrimination mechanism at the cellular level that wouldallow the preferential presentation of PRR-associated antigens.Considering the impact of ROS production in antigen proces-sing, it will be of interest to investigate whether phagosomalNOX2 and the different Rac isoforms are regulated autono-mously, and if this is modulated by TLR signaling (Fig. 7).

Closing Remarks

NOX2 is known to play a major role in innate immunityfor several decades. In the last few years, however, it hasbecome clear that NOX2 is also involved in the establish-ment of adaptive immunity. Its role in the control ofphagosomal pH, and thereby of the efficacy of antigencross-presentation in DCs, represents a clear illustration ofhow NOX2 can influence CD8 + T cell responses. Interest-ingly, CGD patients do occasionally develop autoimmunepathologies, suggesting again a role for NOX2 in adaptiveresponses. In the next years, it will be interesting to unravelpossible mechanisms involved and to find other possibleconnections between NOX family members and adaptiveimmune responses.

References

1. Abo A, Pick E, Hall A, Totty N, Teahan CG, and Segal AW.Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature 353: 668–670, 1991.

2. Allen LA, Beecher BR, Lynch JT, Rohner OV, and WittineLM. Helicobacter pylori disrupts NADPH oxidase target-ing in human neutrophils to induce extracellular superox-ide release. J Immunol 174: 3658–3667, 2005.

3. Ambruso DR, Cusack N, and Thurman G. NADPH oxidaseactivity of neutrophil specific granules: requirements forcytosolic components and evidence of assembly during cellactivation. Mol Genet Metab 81: 313–321, 2004.

4. Amigorena S and Savina A. Intracellular mechanisms ofantigen cross presentation in dendritic cells. Curr OpinImmunol 22: 109–117, 2010.

5. Bachem A, Guttler S, Hartung E, Ebstein F, Schaefer M,Tannert A, Salama A, Movassaghi K, Opitz C, Mages HW,Henn V, Kloetzel PM, Gurka S, and Kroczek RA. Superiorantigen cross-presentation and XCR1 expression definehuman CD11c + CD141 + cells as homologues of mouseCD8 + dendritic cells. J Exp Med 207: 1273–1281, 2010.

724 KOTSIAS ET AL.

6. Balce DR, Li B, Allan ER, Rybicka JM, Krohn RM, andYates RM. Alternative activation of macrophages by IL-4enhances the proteolytic capacity of their phagosomesthrough synergistic mechanisms. Blood 118: 4199–4208, 2011.

7. Basha G, Omilusik K, Chavez-Steenbock A, Reinicke AT,Lack N, Choi KB, and Jefferies WA. A CD74-dependentMHC class I endolysosomal cross-presentation pathway.Nat Immunol 13: 237–245, 2012.

8. Bedard K and Krause KH. The NOX family of ROS-gen-erating NADPH oxidases: physiology and pathophysi-ology. Physiol Rev 87: 245–313, 2007.

9. Berendes H, Bridges RA, and Good RA. A fatal granulo-matosus of childhood: the clinical study of a new syn-drome. Minn Med 40: 309–312, 1957.

10. Berton G, Bellavite P, de Nicola G, Dri P, Rossi F.Berton G,Bellavite P, de Nicola G, Dri P, and Rossi F. Plasmamembrane and phagosome localisation of the activatedNADPH oxidase in elicited peritoneal macrophages of theguinea-pig. J Pathol 136: 241–252, 1982.

11. Bienert GP, Schjoerring JK, and Jahn TP. Membranetransport of hydrogen peroxide. Biochim Biophys Acta 1758:994–1003, 2006.

12. Biswas S, Chida AS, and Rahman I. Redox modifications ofprotein-thiols: emerging roles in cell signaling. BiochemPharmacol 71: 551–564. 2006.

13. Bjorgvinsdottir H, Zhen L, and Dinauer MC. Cloning ofmurine gp91phox cDNA and functional expression in ahuman X-linked chronic granulomatous disease cell line.Blood 87: 2005–2010, 1996.

14. Blander JM and Medzhitov R. Toll-dependent selection ofmicrobial antigens for presentation by dendritic cells. Nat-ure 440: 808–812, 2006.

15. Blander JM. Signalling and phagocytosis in the orchestra-tion of host defence. Cell Microbiol 9: 290–299, 2007.

16. Bokoch GM and Zhao T. Regulation of the phagocyteNADPH oxidase by Rac GTPase. Antioxid Redox Signal 8:1533–1548, 2006.

17. Bonnefoy-Berard N, Munshi A, Yron I, Wu S, Collins TL,Deckert M, Shalom-Barak T, Giampa L, Herbert E, Her-nandez J, Meller N, Couture C, and Altman A. Vav: func-tion and regulation in hematopoietic cell signaling. StemCells 14: 250–268, 1996.

18. Borregaard N, Heiple JM, Simons ER, and Clark RA. Sub-cellular localization of the b-cytochrome component of thehuman neutrophil microbicidal oxidase: translocationduring activation. J Cell Biol 97: 52–61, 1983.

19. Bridges RA, Berendes H, and Good RA. A fatal granulo-matous disease of childhood; the clinical, pathological, andlaboratory features of a new syndrome. AMA J Dis Child 97:387–408, 1959.

20. Burgdorf S and Kurts C. Endocytosis mechanisms and thecell biology of antigen presentation. Curr Opin Immunol 20:89–95, 2008.

21. Carbone FR and Bevan MJ. Class I-restricted processingand presentation of exogenous cell-associated antigenin vivo. J Exp Med 171: 377–387, 1990.

22. Casbon AJ, Allen LA, Dunn KW, and Dinauer MC. Mac-rophage NADPH oxidase flavocytochrome B localizes tothe plasma membrane and Rab11-positive recycling endo-somes. J Immunol 182: 2325–2339, 2009.

23. Cathcart MK. Regulation of superoxide anion productionby NADPH oxidase in monocytes/macrophages: contri-butions to atherosclerosis. Arterioscler Thromb Vasc Biol 24:23–28, 2004.

24. Cebrian I, Visentin G, Blanchard N, Jouve M, Bobard A,Moita C, Enninga J, Moita LF, Amigorena S, and SavinaA. Sec22b regulates phagosomal maturation and anti-gen crosspresentation by dendritic cells. Cell 147: 1355–1368, 2011.

25. Chemali M, Radtke K, Desjardins M, and English L. Al-ternative pathways for MHC class I presentation: a newfunction for autophagy. Cell Mol Life Sci 68: 1533–1541,2011.

26. Cheng G, Ritsick D, and Lambeth JD. Nox3 regulationby NOXO1, p47phox, and p67phox. J Biol Chem 279: 34250–34255, 2004.

27. Chiang E, Dang O, Anderson K, Matsuzawa A, Ichijo H,and David M. Cutting edge: apoptosis-regulating signalkinase 1 is required for reactive oxygen species-mediatedactivation of IFN regulatory factor 3 by lipopolysaccharide.J Immunol 176: 5720–5724, 2006.

28. Clark RA, Volpp BD, Leidal KG, and Nauseef WM. Twocytosolic components of the human neutrophil respiratoryburst oxidase translocate to the plasma membrane duringcell activation. J Clin Invest 85: 714–721, 1990.

29. Crespo P, Schuebel KE, Ostrom AA, Gutkind JS, and Bus-telo XR. Phosphotyrosine-dependent activation of Rac-1GDP/GTP exchange by the vav proto-oncogene product.Nature 385: 169–172, 1997.

30. Cresswell P, Ackerman AL, Giodini A, Peaper DR, andWearsch PA. Mechanisms of MHC class I-restricted antigenprocessing and cross-presentation. Immunol Rev 207: 145–157, 2005.

31. Crotzer VL and Blum JS. Autophagy and its role in MHC-mediated antigen presentation. J Immunol 182: 3335–3341, 2009.

32. del Rio ML, Rodriguez-Barbosa JI, Kremmer E, and ForsterR. CD103- and CD103 + bronchial lymph node dendriticcells are specialized in presenting and cross-presenting in-nocuous antigen to CD4 + and CD8 + T cells. J Immunol178: 6861–6866, 2007.

33. Delamarre L, Couture R, Mellman I, and Trombetta ES.Enhancing immunogenicity by limiting susceptibility tolysosomal proteolysis. J Exp Med 203: 2049–2055, 2006.

34. Delamarre L, Pack M, Chang H, Mellman I, and Trom-betta ES. Differential lysosomal proteolysis in antigen-presenting cells determines antigen fate. Science 307:1630–1634, 2005.

35. Desch AN, Randolph GJ, Murphy K, Gautier EL, Kedl RM,Lahoud MH, Caminschi I, Shortman K, Henson PM, andJakubzick CV. CD103 + pulmonary dendritic cells prefer-entially acquire and present apoptotic cell-associated anti-gen. J Exp Med 208: 1789–1797, 2011.

36. Desjardins M, Huber LA, Parton RG, and Griffiths G. Bio-genesis of phagolysosomes proceeds through a sequentialseries of interactions with the endocytic apparatus. J CellBiol 124: 677–688, 1994.

37. Diekmann D, Abo A, Johnston C, Segal AW, and Hall A.Interaction of Rac with p67phox and regulation ofphagocytic NADPH oxidase activity. Science 265: 531–533, 1994.

38. Dudziak D, Kamphorst AO, Heidkamp GF, Buchholz VR,Trumpfheller C, Yamazaki S, Cheong C, Liu K, Lee HW,Park CG, Steinman RM, and Nussenzweig MC. Differentialantigen processing by dendritic cell subsets in vivo. Science315: 107–111, 2007.

39. El-Benna J, Dang PM, and Gougerot-Pocidalo MA. Primingof the neutrophil NADPH oxidase activation: role of


p47phox phosphorylation and NOX2 mobilization to theplasma membrane. Semin Immunopathol 30: 279–289, 2008.

40. El-Benna J, Dang PM, Gougerot-Pocidalo MA, Marie JC,and Braut-Boucher F. p47phox, the phagocyte NADPHoxidase/NOX2 organizer: structure, phosphorylation andimplication in diseases. Exp Mol Med 41: 217–225, 2009.

41. Elsen S, Doussiere J, Villiers CL, Faure M, Berthier R, Pa-paioannou A, Grandvaux N, Marche PN, and Vignais PV.Cryptic O2 - -generating NADPH oxidase in dendritic cells.J Cell Sci 117: 2215–2226, 2004.

42. Feng Y and Forgac M. A novel mechanism for regulation ofvacuolar acidification. J Biol Chem 267: 19769–19772, 1992.

43. Feng Y and Forgac M. Inhibition of vacuolar H( + )-ATPaseby disulfide bond formation between cysteine 254 andcysteine 532 in subunit A. J Biol Chem 269: 13224–13230,1994.

44. Forman HJ and Torres M. Redox signaling in macrophages.Mol Aspects Med 22: 189–216, 2001.

45. Forman HJ and Torres M. Reactive oxygen species and cellsignaling: respiratory burst in macrophage signaling. Am JRespir Crit Care Med 166: S4–S8, 2002.

46. Foster CB, Lehrnbecher T, Mol F, Steinberg SM, Venzon DJ,Walsh TJ, Noack D, Rae J, Winkelstein JA, Curnutte JT, andChanock SJ. Host defense molecule polymorphisms influ-ence the risk for immune-mediated complications inchronic granulomatous disease. J Clin Invest 102: 2146–2155, 1998.

47. Garcia RC and Segal AW. Changes in the subcellular dis-tribution of the cytochrome b-245 on stimulation of humanneutrophils. Biochem J 219: 233–242, 1984.

48. Glogauer M, Marchal CC, Zhu F, Worku A, ClausenBE, Foerster I, Marks P, Downey GP, Dinauer M, andKwiatkowski DJ. Rac1 deletion in mouse neutrophils hasselective effects on neutrophil functions. J Immunol 170:5652–5657, 2003.

49. Graham DB, Stephenson LM, Lam SK, Brim K, Lee HM,Bautista J, Gilfillan S, Akilesh S, Fujikawa K, and Swat W.An ITAM-signaling pathway controls cross-presentation ofparticulate but not soluble antigens in dendritic cells. J ExpMed 204: 2889–2897, 2007.

50. Gwinn MR and Vallyathan V. Respiratory burst: role insignal transduction in alveolar macrophages. J Toxicol En-viron Health B Crit Rev 9: 27–39, 2006.

51. Ha EM, Lee KA, Seo YY, Kim SH, Lim JH, Oh BH, Kim J,and Lee WJ. Coordination of multiple dual oxidase-regulatory pathways in responses to commensal and infectiousmicrobes in drosophila gut. Nat Immunol 10: 949–957, 2009.

52. Hampton MB, Kettle AJ, and Winterbourn CC. Inside theneutrophil phagosome: oxidants, myeloperoxidase, andbacterial killing. Blood 92: 3007–3017, 1998.

53. Harada T, Miyake M, and Imai Y. Evasion of Legionellapneumophila from the bactericidal system by reactive ox-ygen species (ROS) in macrophages. Microbiol Immunol 51:1161–1170, 2007.

54. Hastings KT, Lackman RL, and Cresswell P. Functionalrequirements for the lysosomal thiol reductase GILT inMHC class II-restricted antigen processing. J Immunol 177:8569–8577, 2006.

55. Hattori H, Subramanian KK, Sakai J, Jia Y, Li Y, Porter TF,Loison F, Sarraj B, Kasorn A, Jo H, Blanchard C, Zirkle D,McDonald D, Pai SY, Serhan CN, and Luo HR. Small-molecule screen identifies reactive oxygen species as keyregulators of neutrophil chemotaxis. Proc Natl Acad Sci U S A107: 3546–3551, 2010.

56. Holland SM. Chronic granulomatous disease. Clin Rev Al-lergy Immunol 38: 3–10, 2010.

57. Honey K and Rudensky AY. Lysosomal cysteine proteasesregulate antigen presentation. Nat Rev Immunol 3: 472–482,2003.

58. Hsing LC and Rudensky AY. The lysosomal cysteine pro-teases in MHC class II antigen presentation. Immunol Rev207: 229–241, 2005.

59. Huang J, Canadien V, Lam GY, Steinberg BE, Dinauer MC,Magalhaes MA, Glogauer M, Grinstein S, and Brumell JH.Activation of antibacterial autophagy by NADPH oxidases.Proc Natl Acad Sci U S A 106: 6226–6231, 2009.

60. Iyoda T, Shimoyama S, Liu K, Omatsu Y, Akiyama Y,Maeda Y, Takahara K, Steinman RM, and Inaba K. TheCD8 + dendritic cell subset selectively endocytoses dy-ing cells in culture and in vivo. J Exp Med 195: 1289–1302,2002.

61. Jancic C, Savina A, Wasmeier C, Tolmachova T, El-Benna J,Dang PM, Pascolo S, Gougerot-Pocidalo MA, Raposo G,Seabra MC, and Amigorena S. Rab27a regulates phagoso-mal pH and NADPH oxidase recruitment to dendritic cellphagosomes. Nat Cell Biol 9: 367–378, 2007.

62. Joiner KA, Fuhrman SA, Miettinen HM, Kasper LH, andMellman I. Toxoplasma gondii: fusion competence ofparasitophorous vacuoles in Fc receptor-transfected fibro-blasts. Science 249: 641–646, 1990.

63. Jongbloed SL, Kassianos AJ, McDonald KJ, Clark GJ, Ju X,Angel CE, Chen CJ, Dunbar PR, Wadley RB, Jeet V, VulinkAJ, Hart DN, and Radford KJ. Human CD141 + (BDCA-3) + dendritic cells (DCs) represent a unique myeloid DCsubset that cross-presents necrotic cell antigens. J Exp Med207: 1247–1260, 2010.

64. Jung S, Unutmaz D, Wong P, Sano G, De los Santos K,Sparwasser T, Wu S, Vuthoori S, Ko K, Zavala F, PamerEG, Littman DR, and Lang RA. In vivo depletion ofCD11c + dendritic cells abrogates priming of CD8 + T cellsby exogenous cell-associated antigens. Immunity 17: 211–220, 2002.

65. Kao YY, Gianni D, Bohl B, Taylor RM, and Bokoch GM.Identification of a conserved Rac-binding site on NADPHoxidases supports a direct GTPase regulatory mechanism. JBiol Chem 283: 12736–12746, 2008.

66. Karlsson A and Dahlgren C. Assembly and activation ofthe neutrophil NADPH oxidase in granule membranes.Antioxid Redox Signal 4: 49–60, 2002.

67. Kawahara T, Kohjima M, Kuwano Y, Mino H, Teshima-Kondo S, Takeya R, Tsunawaki S, Wada A, Sumimoto H,and Rokutan K. Helicobacter pylori lipopolysaccharideactivates Rac1 and transcription of NADPH oxidase Nox1and its organizer NOXO1 in guinea pig gastric mucosalcells. Am J Physiol Cell Physiol 288: C450–C457, 2005.

68. Kawahara T, Kuwano Y, Teshima-Kondo S, Takeya R,Sumimoto H, Kishi K, Tsunawaki S, Hirayama T, and Ro-kutan K. Role of nicotinamide adenine dinucleotide phos-phate oxidase 1 in oxidative burst response to Toll-likereceptor 5 signaling in large intestinal epithelial cells. JImmunol 172: 3051–3058, 2004.

69. Kawahara T, Ritsick D, Cheng G, and Lambeth JD. Pointmutations in the proline-rich region of p22phox are domi-nant inhibitors of Nox1- and Nox2-dependent reactive ox-ygen generation. J Biol Chem 280: 31859–31869, 2005.

70. Kim YM, Kim KE, Koh GY, Ho Y, and Lee K. Hydrogenperoxide produced by angiopoietin-1 mediates angiogene-sis. Cancer Res 66: 6167–6174, 2006.

726 KOTSIAS ET AL.

71. Kjeldsen L, Sengeløv H, Lollike K, Nielsen MH, andBorregaard N. Isolation and characterization of gelatinasegranules from human neutrophils. Blood 83: 1640–1649,1994.

72. Knaus UG, Heyworth PG, Evans T, Curnutte JT, and Bo-koch GM. Regulation of phagocyte oxygen radical pro-duction by the GTP-binding protein Rac 2. Science 254:1512–1515, 1991.

73. Krause KH and Bedard K. NOX enzymes in immuno-inflammatory pathologies. Semin Immunopathol 30: 193–194, 2008.

74. Kuiper JW, Sun C, Magalhaes MA, and Glogauer M. Racregulates PtdInsP3 signaling and the chemotactic compassthrough a redox-mediated feedback loop. Blood 118: 6164–6171, 2011.

75. Lam GY, Huang J, and Brumell JH. The many roles ofNOX2 NADPH oxidase-derived ROS in immunity. SeminImmunopathol 32: 415–430, 2010.

76. Lambeth JD. NOX enzymes and the biology of reactiveoxygen. Nat Rev Immunol 4: 181–189, 2004.

77. Lapouge K, Smith SJ, Walker PA, Gamblin SJ, Smerdon SJ,and Rittinger K. Structure of the TPR domain of p67phox incomplex with Rac.GTP. Mol Cell 6: 899–907, 2000.

78. Lee SH, Taek Han S, Choi SW, Sung SY, You HJ, Ye SK,and Chung MH. Inhibition of Rac and Rac-linked functionsby 8-oxo-2¢-deoxyguanosine in murine macrophages. FreeRadic Res 43: 78–84, 2009.

79. Leiva N, Pavarotti M, Colombo MI, and Damiani MT.Reconstitution of recycling from the phagosomal com-partment in streptolysin O-permeabilized macrophages:role of Rab11. Exp Cell Res 312: 1843–1855, 2006.

80. Lennon-Dumenil AM, Bakker AH, Maehr R, Fiebiger E,Overkleeft HS, Rosemblatt M, Ploegh HL, and Lagau-driere-Gesbert C. Analysis of protease activity in live anti-gen-presenting cells shows regulation of the phagosomalproteolytic contents during dendritic cell activation. J ExpMed 196: 529–540, 2002.

81. Liu X, Lu L, Yang Z, Palaniyandi S, Zeng R, Gao LY,Mosser DM, Roopenian DC, and Zhu X. The neonatal FcR-mediated presentation of immune-complexed antigen isassociated with endosomal and phagosomal pH and anti-gen stability in macrophages and dendritic cells. J Immunol186: 4674–4686, 2011.

82. Lodge R, Diallo TO, and Descoteaux A. Leishmaniadonovani lipophosphoglycan blocks NADPH oxidaseassembly at the phagosome membrane. Cell Microbiol 8:1922–1931, 2006.

83. Maemura K, Zheng Q, Wada T, Ozaki M, Takao S, AikouT, Bulkley GB, Klein AS, and Sun Z. Reactive oxygenspecies are essential mediators in antigen presentation byKupffer cells. Immunol Cell Biol 83: 336–343, 2005.

84. Mantegazza AR, Savina A, Vermeulen M, Perez L, GeffnerJ, Hermine O, Rosenzweig SD, Faure F, and Amigorena S.NADPH oxidase controls phagosomal pH and antigencross-presentation in human dendritic cells. Blood 112:4712–4722, 2008.

85. Matsue H, Edelbaum D, Shalhevet D, Mizumoto N, YangC, Mummert ME, Oeda J, Masayasu H, and Takashima A.Generation and function of reactive oxygen species indendritic cells during antigen presentation. J Immunol 171:3010–3018, 2003.

86. Matsuzawa A, Saegusa K, Noguchi T, Sadamitsu C, Nish-itoh H, Nagai S, Koyasu S, Matsumoto K, Takeda K, andIchijo H. ROS-dependent activation of the TRAF6-ASK1-

p38 pathway is selectively required for TLR4-mediatedinnate immunity. Nat Immunol 6: 587–592, 2005.

87. Michaelson D, Silletti J, Murphy G, D’Eustachio P, Rush M,and Philips MR. Differential localization of Rho GTPases inlive cells: regulation by hypervariable regions and RhoGDIbinding. J Cell Biol 152: 111–126, 2001.

88. Miletic AV, Graham DB, Montgrain V, Fujikawa K,Kloeppel T, Brim K, Weaver B, Schreiber R, Xavier R, andSwat W. Vav proteins control MyD88-dependent oxidativeburst. Blood 109: 3360–3368, 2007.

89. Ming W, Li S, Billadeau DD, Quilliam LA, and DinauerMC. The Rac effector p67phox regulates phagocyteNADPH oxidase by stimulating Vav1 guanine nucleotideexchange activity. Mol Cell Biol 27: 312–323, 2007.

90. Nagaoka Y, Otsu K, Okada F, Sato K, Ohba Y, Kotani N,and Fujii J. Specific inactivation of cysteine protease-typecathepsin by singlet oxygen generated from naphtha-lene endoperoxides. Biochem Biophys Res Commun 331: 215–223, 2005.

91. Neefjes J, Jongsma ML, Paul P, and Bakke O. Towards asystems understanding of MHC class I and MHC class IIantigen presentation. Nat Rev Immunol 11: 823–836, 2011.

92. Oakley FD, Abbott D, Li Q, and Engelhardt JF. Signalingcomponents of redox active endosomes: the redoxosomes.Antioxid Redox Signal 11: 1313–1333, 2009.

93. Ogier-Denis E, Mkaddem SB, and Vandewalle A. NOXenzymes and Toll-like receptor signaling. Semin Im-munopathol 30: 291–300, 2008.

94. Olsson LM, Lindqvist AK, Kallberg H, Padyukov L,Burkhardt H, Alfredsson L, Klareskog L, and Holmdahl R.A case-control study of rheumatoid arthritis identifies anassociated single nucleotide polymorphism in the NCF4gene, supporting a role for the NADPH-oxidase complex inautoimmunity. Arthritis Res Ther 9: R98, 2007.

95. Paludan C, Schmid D, Landthaler M, Vockerodt M, KubeD, Tuschl T, and Munz C. Endogenous MHC class II pro-cessing of a viral nuclear antigen after autophagy. Science307: 593–596, 2005.

96. Park DW, Baek K, Kim JR, Lee JJ, Ryu SH, Chin BR, andBaek SH. Resveratrol inhibits foam cell formation viaNADPH oxidase 1- mediated reactive oxygen speciesand monocyte chemotactic protein-1. Exp Mol Med 41: 171–179, 2009.

97. Park HS, Chun JN, Jung HY, Choi C, and Bae YS. Role ofNADPH oxidase 4 in lipopolysaccharide-induced proin-flammatory responses by human aortic endothelial cells.Cardiovasc Res 72: 447–455, 2006.

98. Park HS, Jung HY, Park EY, Kim J, Lee WJ, and Bae YS.Cutting edge: direct interaction of TLR4 with NAD(P)Hoxidase 4 isozyme is essential for lipopolysaccharide-in-duced production of reactive oxygen species and activationof NF-kappa B. J Immunol 173: 3589–3593, 2004.

99. Petry A, Weitnauer M, and Gorlach A. Receptor activa-tion of NADPH oxidases. Antioxid Redox Signal 13: 467–487, 2010.

100. Prokopowicz ZM, Arce F, Biedron R, Chiang CL, Ciszek M,Katz DR, Nowakowska M, Zapotoczny S, Marcinkiewicz J,and Chain BM. Hypochlorous acid: a natural adjuvantthat facilitates antigen processing, cross-priming, andthe induction of adaptive immunity. J Immunol 184: 824–835, 2010.

101. Rada B and Leto TL. Oxidative innate immune defenses byNox/Duox family NADPH oxidases. Contrib Microbiol 15:164–168, 2008.


102. Reeves EP, Lu H, Jacobs HL, Messina CG, Bolsover S,Gabella G, Potma EO, Warley A, Roes J, and Segal AW.Killing activity of neutrophils is mediated through activa-tion of proteases by K + flux. Nature 416: 291–297, 2002.

103. Rink J, Ghigo E, Kalaidzidis Y, and Zerial M. Rab conver-sion as a mechanism of progression from early to late en-dosomes. Cell 122: 735–749, 2005.

104. Rock KL, Gamble S, and Rothstein L. Presentation of ex-ogenous antigen with class I major histocompatibilitycomplex molecules. Science 249: 918–921, 1990.

105. Rodriguez A, Regnault A, Kleijmeer M, Ricciardi-Castagnoli P, and Amigorena S. Selective transport ofinternalized antigens to the cytosol for MHC class I pre-sentation in dendritic cells. Nat Cell Biol 1: 362–368, 1999.

106. Russell DG and Yates RM. TLR signalling and phagosomematuration: an alternative viewpoint. Cell Microbiol 9: 849–850, 2007.

107. Russell DG, Vanderven BC, Glennie S, Mwandumba H,and Heyderman RS. The macrophage marches on its pha-gosome: dynamic assays of phagosome function. Nat RevImmunol 9: 594–600, 2009.

108. Rybicka JM, Balce DR, Chaudhuri S, Allan ER, and YatesRM. Phagosomal proteolysis in dendritic cells is modulatedby NADPH oxidase in a pH-independent manner. EMBO J31: 932–944, 2011.

109. Rybicka JM, Balce DR, Khan MF, Krohn RM, and YatesRM. NADPH oxidase activity controls phagosomal prote-olysis in macrophages through modulation of the lumenalredox environment of phagosomes. Proc Natl Acad Sci U S A107: 10496–10501, 2010.

110. Salmeen A and Barford D. Functions and mechanisms ofredox regulation of cysteine-based phosphatases. AntioxidRedox Signal 7: 560–577, 2005.

111. Sareila O, Kelkka T, Pizzolla A, Hultqvist M, and Holm-dahl R. NOX2 complex-derived ROS as immune regulators.Antioxid Redox Signal 15: 2197–2208, 2011.

112. Savina A and Amigorena S. Phagocytosis and antigenpresentation in dendritic cells. Immunol Rev 219: 143–156,2007.

113. Savina A, Jancic C, Hugues S, Guermonprez P, Vargas P,Moura IC, Lennon-Dumenil AM, Seabra MC, Raposo G,and Amigorena S. NOX2 controls phagosomal pH to reg-ulate antigen processing during crosspresentation by den-dritic cells. Cell 126: 205–218, 2006.

114. Savina A, Peres A, Cebrian I, Carmo N, Moita C, HacohenN, Moita LF, and Amigorena S. The small GTPase Rac2controls phagosomal alkalinization and antigen cross-presentation selectively in CD8( + ) dendritic cells. Immunity30: 544–555, 2009.

115. Savina A, Vargas P, Guermonprez P, Lennon AM, andAmigorena S. Measuring pH, ROS production, maturation,and degradation in dendritic cell phagosomes using cyto-fluorometry-based assays. Methods Mol Biol 595: 383–402,2010.

116. Scherz-Shouval R, Shvets E, Fass E, Shorer H, Gil L, andElazar Z. Reactive oxygen species are essential for autop-hagy and specifically regulate the activity of Atg4. EMBO J26: 1749–1760, 2007.

117. Schnorrer P, Behrens GM, Wilson NS, Pooley JL, Smith CM,El-Sukkari D, Davey G, Kupresanin F, Li M, MaraskovskyE, Belz GT, Carbone FR, Shortman K, Heath WR, andVilladangos JA. The dominant role of CD8 + dendritic cellsin cross-presentation is not dictated by antigen capture.Proc Natl Acad Sci U S A 103: 10729–10734, 2006.

118. Schulz O, Pennington DJ, Hodivala-Dilke K, Febbraio M,and Reis e Sousa C. CD36 or alphavbeta3 and alphavbeta5integrins are not essential for MHC class I cross-presenta-tion of cell-associated antigen by CD8 alpha + murinedendritic cells. J Immunol 168: 6057–6065, 2002.

119. Segal AW. How neutrophils kill microbes. Annu Rev Im-munol 23: 197–223, 2005.

120. Segura E and Villadangos JA. Antigen presentation bydendritic cells in vivo. Curr Opin Immunol 21: 105–110, 2009.

121. Segura E, Valladeau-Guilemond J, Donnadieu MH, Sastre-Garau X, Soumelis V, and Amigorena S. Characterizationof resident and migratory dendritic cells in human lymphnodes. J Exp Med 209: 653–660, 2012.

122. Shortman K and Heath WR. The CD8 + dendritic cellsubset. Immunol Rev. 234: 18–31, 2010.

123. Siemsen DW, Kirpotina LN, Jutila MA, and Quinn MT.Inhibition of the human neutrophil NADPH oxidase byCoxiella burnetii. Microbes Infect 11: 671–679, 2009.

124. Singh R and Cresswell P. Defective cross-presentation ofviral antigens in GILT-free mice. Science 328: 1394–1398,2010.

125. Skrzydlewska E, Sulkowska M, Koda M, and Sulkowski S.Proteolytic-antiproteolytic balance and its regulation incarcinogenesis. World J Gastroenterol 11: 1251–1266, 2005.

126. Songsungthong W, Higgins MC, Rolan HG, Murphy JL,and Mecsas J. ROS-inhibitory activity of YopE is requiredfor full virulence of Yersinia in mice. Cell Microbiol 12: 988–1001, 2010.

127. Stow JL, Manderson AP, and Murray RZ. SNAREing im-munity: the role of SNAREs in the immune system. Nat RevImmunol 6: 919–929, 2006.

128. Strassheim D, Asehnoune K, Park JS, Kim JY, He Q, RichterD, Mitra S, Arcaroli J, Kuhn K, and Abraham E. Modula-tion of bone marrow-derived neutrophil signaling byH2O2: disparate effects on kinases, NF-kappaB, and cyto-kine expression. Am J Physiol Cell Physiol 286: C683–C692,2004.

129. Sumimoto H. Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygenspecies. FEBS J 275: 3249–3277, 2008.

130. Suto D, Iuchi Y, Ikeda Y, Sato K, Ohba Y, and Fujii J. In-activation of cysteine and serine proteases by singlet oxy-gen. Arch Biochem Biophys 461: 151–158, 2007.

131. Tassi S, Carta S, Vene R, Delfino L, Ciriolo MR, and Ru-bartelli A. Pathogen-induced interleukin-1beta processingand secretion is regulated by a biphasic redox response. JImmunol 183: 1456–1462, 2009.

132. Thiele L, Merkle HP, and Walter E. Phagocytosis andphagosomal fate of surface-modified microparticles indendritic cells and macrophages. Pharm Res 20: 221–228,2003.

133. Tran KK and Shen H. The role of phagosomal pH on thesize-dependent efficiency of cross-presentation by dendriticcells. Biomaterials 30: 1356–1362, 2009.

134. Trivedi V, Zhang SC, Castoreno AB, Stockinger W, ShiehEC, Vyas JM, Frickel EM, and Nohturfft A. Im-munoglobulin G signaling activates lysosome/phagosomedocking. Proc Natl Acad Sci U S A 103: 18226–18231, 2006.

135. Trombetta ES, Ebersold M, Garrett W, Pypaert M, andMellman I. Activation of lysosomal function during den-dritic cell maturation. Science 299: 1400–1403, 2003.

136. Ueno N, Takeya R, Miyano K, Kikuchi H, and SumimotoH. The NADPH oxidase Nox3 constitutively produces su-peroxide in a p22phox-dependent manner: its regulation by

728 KOTSIAS ET AL.

oxidase organizers and activators. J Biol Chem 280: 23328–23339, 2005.

137. Vazquez-Torres A, Jones-Carson J, Mastroeni P, Ischir-opoulos H, and Fang FC. Antimicrobial actions of theNADPH phagocyte oxidase and inducible nitric oxidesynthase in experimental salmonellosis. I. Effects on mi-crobial killing by activated peritoneal macrophages in vitro.J Exp Med 192: 227–236, 2000.

138. Vazquez-Torres A, Xu Y, Jones-Carson J, Holden DW,Lucia SM, Dinauer MC, Mastroeni P, and Fang FC. Sal-monella pathogenicity island 2-dependent evasion of thephagocyte NADPH oxidase. Science 287: 1655–1658, 2000.

139. Vieira OV, Botelho RJ, and Grinstein S. Phagosome matu-ration: aging gracefully. Biochem J 366: 689–704, 2002.

140. Vieira OV, Bucci C, Harrison RE, Trimble WS, Lanzetti L,Gruenberg J, Schreiber AD, Stahl PD, and Grinstein S.Modulation of Rab5 and Rab7 recruitment to phagosomesby phosphatidylinositol 3-kinase. Mol Cell Biol 23: 2501–2514, 2003.

141. Vremec D, Zorbas M, Scollay R, Saunders DJ, Ardavin CF,Wu L, and Shortman K. The surface phenotype of dendriticcells purified from mouse thymus and spleen: investigationof the CD8 expression by a subpopulation of dendritic cells.J Exp Med 176: 47–58, 1992.

142. Vulcano M, Dusi S, Lissandrini D, Badolato R, Mazzi P,Riboldi E, Borroni E, Calleri A, Donini M, Plebani A,Notarangelo L, Musso T, and Sozzani S. Toll receptor-mediated regulation of NADPH oxidase in human den-dritic cells. J Immunol 173: 5749–5756, 2004.

143. Yamauchi A, Kim C, Li S, Marchal CC, Towe J, AtkinsonSJ, and Dinauer MC. Rac2-deficient murine macrophageshave selective defects in superoxide production andphagocytosis of opsonized particles. J Immunol 173: 5971–5979, 2004.

144. Yates RM and Russell DG. Real-time spectrofluorometricassays for the lumenal environment of the maturing pha-gosome. Methods Mol Biol 445: 311–325, 2008.

145. Yates RM, Hermetter A, and Russell DG. Recording pha-gosome maturation through the real-time, spectrofluoro-metric measurement of hydrolytic activities. Methods MolBiol 531: 157–171, 2009.

146. Yeung T, Gilbert GE, Shi J, Silvius J, Kapus A, and Grin-stein S. Membrane phosphatidylserine regulates surfacecharge and protein localization. Science 319: 210–213, 2008.

147. Zhang X, Shan P, Jiang G, Cohn L, and Lee PJ. Toll-likereceptor 4 deficiency causes pulmonary emphysema. J ClinInvest 116: 3050–3059, 2006.

Address correspondence to:Dr. Ariel Savina

Institut CurieINSERM U932

26 rue d’UlmParis Cedex 05 75248

France

E-mail: [email protected]

Date of first submission to ARS Central, February 6, 2012; dateof final revised submission, July 3, 2012; date of acceptance,July 25, 2012.

Abbreviations Used

APCs¼ antigen-presenting cellsBMDCs¼ bone marrow-derived dendritic cells

CGD¼ chronic granulomatous diseaseDCs¼dendritic cellsDPI¼diphenylene iodonium

DUOX¼dual oxidaseER¼ endoplasmic reticulum

GEFs¼GDP/GTP-exchange factorsIFN¼ interferonLPS¼ lipopolysaccharides

MHC¼major histocompatibility complexNOX¼NADPH oxidaseOVA¼ ovalbumin

PM¼plasma membranePMA¼phorbol-12-myristate-13-acetatePRRs¼pattern recognition receptorsROS¼ reactive oxygen speciesTLR¼ toll-like receptor

TNFa¼ tumor necrosis factor aTRIF¼Toll/IL-1R domain-containing

adaptor-inducing IFN-ßXP¼ cross-presentation


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