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• W2068853695 abstract "To initiate adaptative cytotoxic immune responses, proteolytic peptides derived from phagocytosed antigens are presented by dendritic cells (DCs) to CD8+ T lymphocytes through a process called antigen “crosspresentation.” The partial degradation of antigens mediated by lysosomal proteases in an acidic environment must be tightly controlled to prevent destruction of potential peptides for T cell recognition. We now describe a specialization of the phagocytic pathway of DCs that allows a fine control of antigen processing. The NADPH oxidase NOX2 is recruited to the DC's early phagosomes and mediates the sustained production of low levels of reactive oxygen species, causing active and maintained alkalinization of the phagosomal lumen. DCs lacking NOX2 show enhanced phagosomal acidification and increased antigen degradation, resulting in impaired crosspresentation. Therefore, NOX2 plays a critical role in conferring DCs the ability to function as specialized phagocytes adapted to process antigens rather than kill pathogens. To initiate adaptative cytotoxic immune responses, proteolytic peptides derived from phagocytosed antigens are presented by dendritic cells (DCs) to CD8+ T lymphocytes through a process called antigen “crosspresentation.” The partial degradation of antigens mediated by lysosomal proteases in an acidic environment must be tightly controlled to prevent destruction of potential peptides for T cell recognition. We now describe a specialization of the phagocytic pathway of DCs that allows a fine control of antigen processing. The NADPH oxidase NOX2 is recruited to the DC's early phagosomes and mediates the sustained production of low levels of reactive oxygen species, causing active and maintained alkalinization of the phagosomal lumen. DCs lacking NOX2 show enhanced phagosomal acidification and increased antigen degradation, resulting in impaired crosspresentation. Therefore, NOX2 plays a critical role in conferring DCs the ability to function as specialized phagocytes adapted to process antigens rather than kill pathogens. To initiate most cytotoxic immune responses, dendritic cells (DCs) must present proteolytic peptides derived from pathogens or infected cells to naïve CD8+ T lymphocytes (Banchereau and Steinman, 1998Banchereau J. Steinman R.M. Dendritic cells and the control of immunity.Nature. 1998; 392: 245-252Crossref PubMed Scopus (11831) Google Scholar). Phagocytosed antigens are presented to CD8+ T lymphocytes through a process called “crosspresentation” (Guermonprez et al., 2002Guermonprez P. Valladeau J. Zitvogel L. Thery C. Amigorena S. Antigen presentation and T cell stimulation by dendritic cells.Annu. Rev. Immunol. 2002; 20: 621-667Crossref PubMed Scopus (1340) Google Scholar, Heath et al., 2004Heath W.R. Belz G.T. Behrens G.M. Smith C.M. Forehan S.P. Parish I.A. Davey G.M. Wilson N.S. Carbone F.R. Villadangos J.A. Cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens.Immunol. Rev. 2004; 199: 9-26Crossref PubMed Scopus (578) Google Scholar). Crosspresentation requires antigen transit through endosomes or phagosomes and eventually partial proteolysis before transport to the cytosol and degradation by the proteasome into 8-9 amino acid peptides. These peptides are then translocated into the lumen of the ER or back into specialized mix ER-phagosome compartments and loaded onto major histocompatibility complex (MHC) class I molecules (Cresswell, 2005Cresswell P. Antigen processing and presentation.Immunol. Rev. 2005; 207: 5-7Crossref PubMed Scopus (45) Google Scholar). This initial partial degradation of antigens must be tightly controlled, as it could destroy potential peptides for T cell recognition. In phagocytic cells, protein degradation is mediated by a large family of proteases, often referred to as “lysosomal proteases.” Most lysosomal proteases have an optimal proteolytic activity between pH 5.5 and 6.5 (Claus et al., 1998Claus V. Jahraus A. Tjelle T. Berg T. Kirschke H. Faulstich H. Griffiths G. Lysosomal enzyme trafficking between phagosomes, endosomes, and lysosomes in J774 macrophages. Enrichment of cathepsin H in early endosomes.J. Biol. Chem. 1998; 273: 9842-9851Crossref PubMed Scopus (162) Google Scholar). Phagosomes fuse first with early and late endosomes and then with lysosomes, thus acquiring progressively both the acidification machinery and the lysosomal proteases from the endocytic pathway (Claus et al., 1998Claus V. Jahraus A. Tjelle T. Berg T. Kirschke H. Faulstich H. Griffiths G. Lysosomal enzyme trafficking between phagosomes, endosomes, and lysosomes in J774 macrophages. Enrichment of cathepsin H in early endosomes.J. Biol. Chem. 1998; 273: 9842-9851Crossref PubMed Scopus (162) Google Scholar, Kjeken et al., 2004Kjeken R. Egeberg M. Habermann A. Kuehnel M. Peyron P. Floetenmeyer M. Walther P. Jahraus A. Defacque H. Kuznetsov S.A. Griffiths G. Fusion between phagosomes, early and late endosomes: a role for actin in fusion between late, but not early endocytic organelles.Mol. Biol. Cell. 2004; 15: 345-358Crossref PubMed Scopus (92) Google Scholar). In macrophages, phagosomes acidify very efficiently, reaching pH 5 in the first 30 min of phagocytosis (Hackam et al., 1998Hackam D.J. Rotstein O.D. Zhang W. Gruenheid S. Gros P. Grinstein S. Host resistance to intracellular infection: mutation of natural resistance-associated macrophage protein 1 (Nramp1) impairs phagosomal acidification.J. Exp. Med. 1998; 188: 351-364Crossref PubMed Scopus (169) Google Scholar, Yates and Russell, 2005Yates R.M. Russell D.G. Phagosome maturation proceeds independently of stimulation of toll-like receptors 2 and 4.Immunity. 2005; 23: 409-417Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Acidification results in a strong activation of lysosomal proteases, effective microbe toxicity, and protein degradation into amino acids. Acidification is mainly but not uniquely mediated by the vacuolar ATPase (V-ATPase), which translocates protons from the cytosol into the lumen of endosomes, lysosomes, and phagosomes. Protons, however, can also enter phagosomes passively, through conductive channels (DeCoursey et al., 2001DeCoursey T.E. Cherny V.V. Morgan D. Katz B.Z. Dinauer M.C. The gp91phox component of NADPH oxidase is not the voltage-gated proton channel in phagocytes, but it helps.J. Biol. Chem. 2001; 276: 36063-36066Crossref PubMed Scopus (61) Google Scholar, Nanda et al., 1994Nanda A. Curnutte J.T. Grinstein S. Activation of H+ conductance in neutrophils requires assembly of components of the respiratory burst oxidase but not its redox function.J. Clin. Invest. 1994; 93: 1770-1775Crossref PubMed Scopus (31) Google Scholar) or through other transporters such as voltage-gated proton channels (DeCoursey et al., 2003DeCoursey T.E. Morgan D. Cherny V.V. The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels.Nature. 2003; 422: 531-534Crossref PubMed Scopus (243) Google Scholar). In neutrophils, another large protein complex, the NADPH oxidase NOX2, mediates the transfer of electrons across endocytic (and plasma) membranes and also influences the phagosomal pH. This multicomponent enzyme is unassembled and inactive in resting cells but assembles at the phagosomal or plasma membrane in a stimulus-dependent manner (El-Benna et al., 2005El-Benna J. Dang P.M. Gougerot-Pocidalo M.A. Elbim C. Phagocyte NADPH oxidase: a multicomponent enzyme essential for host defenses.Arch. Immunol. Ther. Exp. (Warsz.). 2005; 53: 199-206PubMed Google Scholar, Segal, 2005Segal A.W. How neutrophils kill microbes.Annu. Rev. Immunol. 2005; 23: 197-223Crossref PubMed Scopus (1167) Google Scholar). Oxidase subunits include cytosolic proteins such as p47phox, p67phox, p40phox, and rac1 or rac2 (depending on the phagocyte cell type) and an integral membrane heterodimer, the cytochrom b558 (cyt b558), composed of p22phox and gp91phox. NOX2 activity generates superoxide anions in the phagocytic lumen, which dismutate to produce hydrogen peroxide and other reactive oxygen species (ROS). Production of ROS in neutrophils therefore consumes important amounts of protons, causing a transient but strong alkalinization (reaching pH 8) of the phagosome lumen during the oxidative burst (Jiang et al., 1997Jiang Q. Griffin D.A. Barofsky D.F. Hurst J.K. Intraphagosomal chlorination dynamics and yields determined using unique fluorescent bacterial mimics.Chem. Res. Toxicol. 1997; 10: 1080-1089Crossref PubMed Scopus (73) Google Scholar, Segal et al., 1981Segal A.W. Geisow M. Garcia R. Harper A. Miller R. The respiratory burst of phagocytic cells is associated with a rise in vacuolar pH.Nature. 1981; 290: 406-409Crossref PubMed Scopus (272) Google Scholar). Very rapidly, however, ROS production stops, phagosome acidification resumes, and the pH drops within 30 min of phagocytosis (Segal, 2005Segal A.W. How neutrophils kill microbes.Annu. Rev. Immunol. 2005; 23: 197-223Crossref PubMed Scopus (1167) Google Scholar, Segal et al., 1981Segal A.W. Geisow M. Garcia R. Harper A. Miller R. The respiratory burst of phagocytic cells is associated with a rise in vacuolar pH.Nature. 1981; 290: 406-409Crossref PubMed Scopus (272) Google Scholar). DCs also express NOX2 but at relatively low levels (around 5% of the levels found in neutrophils) (Elsen et al., 2004Elsen S. Doussiere J. Villiers C.L. Faure M. Berthier R. Papaioannou A. Grandvaux N. Marche P.N. Vignais P.V. Cryptic O2- -generating NADPH oxidase in dendritic cells.J. Cell Sci. 2004; 117: 2215-2226Crossref PubMed Scopus (47) Google Scholar). The very low levels of ROS production in DCs suggest that NOX2 may not be involved in microbe killing, particularly since DCs' main function is mostly related to the initiation of adaptative immune responses and not to microbe toxicity. Indeed, DCs have developed specific means of control of endocytic functions that allow efficient antigen presentation. These specializations of the endocytic pathway include the regulated transport of MHC molecules (Mellman and Steinman, 2001Mellman I. Steinman R.M. Dendritic cells: specialized and regulated antigen processing machines.Cell. 2001; 106: 255-258Abstract Full Text Full Text PDF PubMed Scopus (1746) Google Scholar, Pierre et al., 1997Pierre P. Turley S.J. Gatti E. Hull M. Meltzer J. Mirza A. Inaba K. Steinman R.M. Mellman I. Developmental regulation of MHC class II transport in mouse dendritic cells.Nature. 1997; 388: 787-792Crossref PubMed Scopus (632) Google Scholar), the transport of antigen from endosomes and phagosomes into the cytosol (Guermonprez and Amigorena, 2005Guermonprez P. Amigorena S. Pathways for antigen crosspresentation.Springer Semin. Immunopathol. 2005; 26: 257-271Crossref PubMed Scopus (74) Google Scholar), and the recruitment of ER proteins to phagosomes (Cresswell, 2005Cresswell P. Antigen processing and presentation.Immunol. Rev. 2005; 207: 5-7Crossref PubMed Scopus (45) Google Scholar, Guermonprez et al., 2003Guermonprez P. Saveanu L. Kleijmeer M. Davoust J. Van Endert P. Amigorena S. ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells.Nature. 2003; 425: 397-402Crossref PubMed Scopus (594) Google Scholar, Houde et al., 2003Houde M. Bertholet S. Gagnon E. Brunet S. Goyette G. Laplante A. Princiotta M.F. Thibault P. Sacks D. Desjardins M. Phagosomes are competent organelles for antigen cross-presentation.Nature. 2003; 425: 402-406Crossref PubMed Scopus (582) Google Scholar). Recent results show that DCs bear low levels of lysosomal proteases (Delamarre et al., 2005Delamarre L. Pack M. Chang H. Mellman I. Trombetta E.S. Differential lysosomal proteolysis in antigen-presenting cells determines antigen fate.Science. 2005; 307: 1630-1634Crossref PubMed Scopus (535) Google Scholar, Lennon-Dumenil et al., 2002Lennon-Dumenil A.M. Bakker A.H. Maehr R. Fiebiger E. Overkleeft H.S. Rosemblatt M. Ploegh H.L. Lagaudriere-Gesbert C. Analysis of protease activity in live antigen-presenting cells shows regulation of the phagosomal proteolytic contents during dendritic cell activation.J. Exp. Med. 2002; 196: 529-540Crossref PubMed Scopus (171) Google Scholar) and that lysosome acidification in immature DCs is inefficient due to limited recruitment of the cytosolic subunits of the V-ATPase to endosomes and lysosomes, as compared to macrophages or mature DCs (Trombetta et al., 2003Trombetta E.S. Ebersold M. Garrett W. Pypaert M. Mellman I. Activation of lysosomal function during dendritic cell maturation.Science. 2003; 299: 1400-1403Crossref PubMed Scopus (529) Google Scholar). DCs' endocytic pathway can therefore be considered as “specialized” antigen presentation compartments rather than purely degradative organelles. To date, however, none of these specializations of the endocytic compartments in DCs was shown genetically to be indispensable for antigen presentation to T cells. We now describe a novel specific adaptation of DCs' endocytic pathway to the antigen presentation function. NOX2, a major player of innate immunity in neutrophils, is recruited to immature DC phagosomes, causing active and sustained phagosome alkalinization. In NOX2-defective DCs, phagosomal acidification and antigen degradation were increased, causing a defect in crosspresentation. These results provide the first genetic evidence that a specialization of DCs' endocytic pathway, i.e., active alkalinization of phagosomes by NOX2, is required for efficient antigen crosspresentation to CD8+ T cells. Efficient antigen processing in phagosomes requires the limited and controlled degradation of protein antigens from phagocytosed cells or cell fragments. Generation of the maximal possible array of peptides for loading on MHC molecules without destroying potentially antigenic peptides implicates a precise control of the activity of lysosomal proteases. One of the most direct ways of controlling the activity of lysosomal proteases is certainly by the pH. In order to measure phagosomal pH in DCs accurately, we set up phagosomal pH measurement by flow cytometry (fluorescence activated cell sorter, or FACS). Latex beads were coated with pH-sensitive (fluorescein isothiocyanate, or FITC) and pH-insensitive (FluoProbe647) fluorescent dyes. After different times of phagocytosis of the beads, the fluorescence intensity was quantified using FACS. The ratio in fluorescence intensity between the two dyes reflected the pH in the phagosomal environment, as shown by imposing fixed pH after permeabilization of the cells (Figure S1A). As shown in Figure 1A, after a 20 min phagocytosis pulse and 30 min of incubation at 37°C (chase), the fluorescence intensity of FITC was much higher in DCs than in macrophage cell line RAW 264.7, indicating that the pH in DC phagosomes was less acidic than in macrophages. By reporting the mean fluorescence intensity in the two cell populations to a standard curve (see Figure S1 and the Experimental Procedures), the actual pH values in phagosomes were determined. As expected, the pH in bone marrow-derived macrophages (BMMO) and RAW 264.7 (Figure 1B) or J774 cells (not shown) was below 6 after a 20 min pulse and a 10 min chase and acidified further over a 3 hr chase, reaching values around pH 5. In contrast, the pH in DC phagosomes was much higher, reaching 7.5 after 60–120 min (Figure 1B). No significant acidification was observed during the first 3 hr of phagocytosis, although the pH did gradually acidify after longer times of phagocytosis (not shown). A similarly alkaline phagosomal pH (around 7.3) was also found in freshly isolated spleen DCs (Figure 1C). Therefore, the pH in DC phagosomes is alkaline and does not acidify significantly in the first 3 hr of phagocytosis. The previously described ineffective acidification in DC lysosomes (Trombetta et al., 2003Trombetta E.S. Ebersold M. Garrett W. Pypaert M. Mellman I. Activation of lysosomal function during dendritic cell maturation.Science. 2003; 299: 1400-1403Crossref PubMed Scopus (529) Google Scholar) is probably insufficient to account for these results since the phagosomal pH reached values actually higher than the extracellular medium, suggesting the existence of an active mechanism of alkalinization. We reasoned that if such an alkalinization process existed in DCs, it should become more evident upon blockade of the V-ATPase present in DC phagosomes. This hypothesis was addressed by analyzing DCs' phagosomal pH in the presence of ConcanamycinB (ConB, a specific inhibitor of the V-ATPase) (Benaroch et al., 1995Benaroch P. Yilla M. Raposo G. Ito K. Miwa K. Geuze H.J. Ploegh H.L. How MHC class II molecules reach the endocytic pathway.EMBO J. 1995; 14: 37-49Crossref PubMed Scopus (148) Google Scholar, Yilla et al., 1993Yilla M. Tan A. Ito K. Miwa K. Ploegh H.L. Involvement of the vacuolar H(+)-ATPases in the secretory pathway of HepG2 cells.J. Biol. Chem. 1993; 268: 19092-19100Abstract Full Text PDF PubMed Google Scholar). As shown in Figure 1D, 1.5 nM of ConB induced a strong alkalinization of the phagosomal pH, reaching pH 8 (the higher limit of the FITC-based pH measure system used here) after 10 min of chase. In order to explore if such phagosomal alkalinization system was specific to DCs, we performed similar experiments using BMMO. Low (1.5 nM) ConB concentrations had no significant effect on phagosomal pH in macrophages, while high concentrations (30 nM) neutralized the phagosomal pH (from pH 5.3–5.8 to pH 7–7.35) (Figure 1E). As shown previously (Hackam et al., 1998Hackam D.J. Rotstein O.D. Zhang W. Gruenheid S. Gros P. Grinstein S. Host resistance to intracellular infection: mutation of natural resistance-associated macrophage protein 1 (Nramp1) impairs phagosomal acidification.J. Exp. Med. 1998; 188: 351-364Crossref PubMed Scopus (169) Google Scholar, Yates and Russell, 2005Yates R.M. Russell D.G. Phagosome maturation proceeds independently of stimulation of toll-like receptors 2 and 4.Immunity. 2005; 23: 409-417Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), alkalinization of the phagosomal pH was observed in BMMO upon blockade of the V-ATPase. We concluded that DCs and not macrophages bear an active machinery of phagosomal alkalinization, which maintains the phagosomal pH between 7 and 7.5 in the first few hours after phagocytosis. Upon inactivation of the V-ATPase, the phagosomal pH in DCs (but not in BMMO) alkalinizes strongly, showing that the V-ATPase is active, at least to some extent, in DC phagosomes. In neutrophils, NOX2 generates ROS, which causes transient phagosome alkalinization, in part through the consumption of protons in the phagocytic lumen (Lee et al., 2003Lee W.L. Harrison R.E. Grinstein S. Phagocytosis by neutrophils.Microbes Infect. 2003; 5: 1299-1306Crossref PubMed Scopus (233) Google Scholar, Segal, 2005Segal A.W. How neutrophils kill microbes.Annu. Rev. Immunol. 2005; 23: 197-223Crossref PubMed Scopus (1167) Google Scholar). We therefore hypothesized that ROS generation by NOX2 in DC phagosomes could be responsible for the active alkalinization of phagosomes in DCs. Since the intracellular localization of NOX2 transmembrane subunits in DCs has not been previously examined, we first analyzed the subcellular distribution of gp91phox using immunofluorescence and confocal microscopy. In resting DCs, gp91phox localizes to vesicular structures (Figure 2A) distributed throughout the cell, including dendrites, suggesting proximity with the plasma membrane. To investigate the morphology of gp91phox-positives structures, we analyzed the intracellular stores of cyt b558 using immunoelectron microscopy. Specific labeling of dense structures, similar to cyt b558-containing vesicles in neutrophils (Calafat et al., 1993Calafat J. Kuijpers T.W. Janssen H. Borregaard N. Verhoeven A.J. Roos D. Evidence for small intracellular vesicles in human blood phagocytes containing cytochrome b558 and the adhesion molecule CD11b/CD18.Blood. 1993; 81: 3122-3129Crossref PubMed Google Scholar), was often observed in the cell profiles (Figure 2B). When we analyzed the cells after 30 min of phagocytosis, cyt b558 was also visible in phagosomes (Figure 2C), indicating the recruitment of NOX2 to this compartment. To explore the recruitment of NOX2 to phagosomes more precisely, phagosomes were purified from wt and gp91phox-deficient DCs after different times of phagocytosis. DCs from gp91phox−/− mice developed normally, displayed a phenotype indistinguishable from that of wt (wild-type) DCs, and responded normally to LPS (Figure S2). Recruitment of the gp91phox to purified phagosomes was analyzed by Western blot. As shown in Figure 2D, gp91phox was recruited to phagosomes over time from wt DCs but not from gp91phox-deficient DCs. Quantification of the Western blots showed that the membrane components of NOX2 are effectively and rapidly recruited to DC phagosomes (Figure 2E). We next attempted to determine if NOX2 was active in DC phagosomes. Activation of NOX2 implicates the recruitment of the cytosolic subunits such as p47phox to the membrane subunits (Cross and Segal, 2004Cross A.R. Segal A.W. The NADPH oxidase of professional phagocytes-prototype of the NOX electron transport chain systems.Biochim. Biophys. Acta. 2004; 1654: 1-22Crossref PubMed Scopus (351) Google Scholar). A clear label of the phagosomal membrane with anti-p47phox antibodies was evident after 1 hr of phagocytosis in wt DCs (Figure 3A, upper panels). A quantification of the fluorescence level around the phagosomes is depicted in Figure 3B. In gp91phox−/− DCs, no labeling of the phagosomes was observed, consistent with the known role of gp91phox in the membrane recruitment of p47phox (Figures 3A and 3B, lower panels). We conclude that NOX2 is effectively assembled on DC phagosomes only when gp91phox is present, suggesting that NOX2 is functional in this compartment in DCs. DCs have previously been shown to produce low levels of ROS (Elsen et al., 2004Elsen S. Doussiere J. Villiers C.L. Faure M. Berthier R. Papaioannou A. Grandvaux N. Marche P.N. Vignais P.V. Cryptic O2- -generating NADPH oxidase in dendritic cells.J. Cell Sci. 2004; 117: 2215-2226Crossref PubMed Scopus (47) Google Scholar, Matsue et al., 2003Matsue H. Edelbaum D. Shalhevet D. Mizumoto N. Yang C. Mummert M.E. Oeda J. Masayasu H. Takashima A. Generation and function of reactive oxygen species in dendritic cells during antigen presentation.J. Immunol. 2003; 171: 3010-3018Crossref PubMed Scopus (198) Google Scholar). To address the possible generation of ROS specifically in phagosomes, we covalently linked dihydrorhodamine 123 (DHR), a dye that only emits fluorescence under oxidative conditions, to latex beads (Vowells et al., 1995Vowells S.J. Sekhsaria S. Malech H.L. Shalit M. Fleisher T.A. Flow cytometric analysis of the granulocyte respiratory burst: a comparison study of fluorescent probes.J. Immunol. Methods. 1995; 178: 89-97Crossref PubMed Scopus (377) Google Scholar). As shown in Figures 3C (fluorescence microscopy) and 3D (FACS analysis), DHR-coated beads became fluorescent after phagocytosis, showing the production of ROS in DC phagosomes. This fluorescent signal was strongly inhibited by diphenylene iodonium (DPI), a specific inhibitor of flavin-containing enzymes such as NOX2. ROS production was not observed in phagosomes from gp91phox-deficient DCs. We conclude that the phagosomal environment in DCs is oxidative due to ROS generation by NOX2. Like DCs, macrophages have been shown previously to produce ROS during phagocytosis or after activation (Forman and Torres, 2002Forman H.J. Torres M. Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling.Am. J. Respir. Crit. Care Med. 2002; 166: S4-S8Crossref PubMed Scopus (656) Google Scholar). To compare ROS production over time in DCs and macrophages, we used a conventional luminol-based technique for ROS quantification. We first measured ROS production in DCs and BMMO treated with phorbol myristate acetate (PMA). PMA activates the protein kinase C, a potent activator of NOX2 (el Benna et al., 1994el Benna J. Faust L.P. Babior B.M. The phosphorylation of the respiratory burst oxidase component p47phox during neutrophil activation. Phosphorylation of sites recognized by protein kinase C and by proline-directed kinases.J. Biol. Chem. 1994; 269: 23431-23436Abstract Full Text PDF PubMed Google Scholar). ROS production in response to PMA was reproducibly higher and lasted longer in DCs than in macrophages (Figure 4A). In DCs, the inactivation was much slower, and significant ROS production was detected even after 2 hr of stimulation (Figure 4A). As expected, no ROS production was observed in gp91phox-deficient DCs (not shown). We concluded that the total capacity of ROS production is higher and NOX2 inactivation is slower in immature DCs than in BMMO. In order to evaluate ROS production selectively in phagosomes, we modified the classical technique used in Figure 4A by covalently attaching luminol to latex beads. ROS production in DC phagosomes was stronger and more sustained than in phagosomes from BMMO both in the absence (Figure 4B) and the presence of PMA (Figure 4C). In order to exclude that BMMO degrade the luminol attached to the beads, thus preventing efficient ROS detection in phagosomes, PMA was added to macrophages after 3 hr of phagocytosis (Figure 4B, arrow). The activation of NOX2 in phagosomes was also estimated through the analysis of the recruitment of p47phox to phagosomes using immunofluorescence and confocal microscopy. After 30 min of phagocytosis, effective NOX2 assembly on phagosomes was detected in both DCs and BMMO (Figure 4D). The fluorescent labeling of p47phox was more evident after 60 min of chase in both cell types. As shown in Figure 4D (lower panels), after 180 min of phagocytosis, however, only DC phagosomes maintained a clear labeling for p47phox (78% of DC phagosomes were visibly labeled, against 14% of BMMO phagosomes; at least 50 phagosomes analyzed in each case). We concluded that the sustained production of ROS in phagosomes from immature DCs is due to a prolonged assembly and activation of the NOX2 complex in DC phagosomes, as compared to BMMO phagosomes. In order to test the possible involvement of NOX2 in the control of the phagosomal pH, we next measured phagosomal pH in gp91phox −/− DCs. The phagosomal pH was strongly decreased in gp91phox-defective DCs, as compared to wt DCs (Figures 5A and 5B), indicating that NOX2 activity controls pH in DC phagosomes. In contrast, wt and gp91phox-defective BMMO acidify their phagosomes with similar efficiencies (Figure 5C), indicating that the role of NOX2 in the control of phagosomal pH is restricted to DCs. Nevertheless, even in the absence of gp91phox, phagosome acidification in DCs was not as efficient as that observed in macrophages (in which the pH dropped to 5.2 in 60 min; see Figures 1B and 5C). Upon blockade of the V-ATPase with ConB, the marked alkalinization observed in phagosomes from wt DCs was not observed in gp91phox-defective DCs (Figure 5D), showing that NOX2 mediates phagosome alkalinization in DCs. Surprisingly, neutralization of the phagosomal pH by ConB in the gp91phox-deficient DCs was observed only after 120 min, and not after 30 min of phagocytosis (Figure 5D), suggesting that in the long-term absence of NOX2 in phagosomes, other proton import pathways may become preeminent and mediate some degree of acidification. Indeed, when NOX2 was blocked at 30 min chase using DPI in wt DCs, the pH acidified within 15 min (Figure 5E). Upon inactivation of the V-ATPase by ConB, however, the pH increased in 15 min (Figure 5E), showing that in the absence of active NOX2, the V-ATPase still mediated acidification in DC phagosomes. We conclude that NOX2 recruitment to DC phagosomes contributes to alkalinize the phagosomal lumen and maintains the pH above 7, in spite of the activity of the V-ATPase. In the absence of ROS generation in DC phagosomes (in the gp91phox-defective DCs or in the presence of DPI), the pH acidifies rapidly. We next sought to evaluate the functional consequences of such a drop in phagosomal pH in terms of antigen degradation. To follow antigen degradation selectively in phagosomes, as opposed to all endocytic compartments, we set up a quantitative cytofluorometric assay for phagosomal degradation. After phagocytosis of beads covalently attached to OVA, the cells were lysed and the amount of OVA remaining on the beads was quantified using polyclonal OVA-specific antibodies and FACS analysis on the isolated beads. As shown in Figure 6A, a marked decrease over time in the amount of OVA attached to the beads was observed in wt DCs after 20 min pulse and 2 hr chase. This decrease was less pronounced in the presence of the protease inhibitors leupeptin and pepstatin (Figure 6A, lower panel) or in DC phagosomes from Cathepsin S (Cat S)-deficient mice (Figure 6B). We conclude that decreased immunofluorescence on the beads was due to actual degradation rather than denaturation of OVA. As shown in Figure 6C, ConB induced a marked inhibition of OVA degradation, showing that the lysosomal proteases present in phagosomes are more active at pH 7–7.5 (the pH in control DCs) than at pH 7.5–8 (the pH in ConB-treated DCs; see Figure 1D). On the contrary, in gp91phox-deficient DCs, in which the phagosomal pH is more acidic than in wt DCs (see Figure 5), the degradation of bead bound OVA was accelerated as compared to wt DCs, as shown in fluorescence intensity histograms from one representative experiment after 30 and 120 min of chase (Figure 6D). Over five independent experiments, the acceleration of OVA degradation in DCs from gp91phox-deficient mice, as compared to wt DCs, was most evident after 30 min of chase (Figure 6E). We conclude that NOX2 activity in DC phagosomes limits antigen degradat" @default.
• W2068853695 created "2016-06-24" @default.
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• W2068853695 date "2006-07-01" @default.
• W2068853695 modified "2023-10-18" @default.
• W2068853695 title "NOX2 Controls Phagosomal pH to Regulate Antigen Processing during Crosspresentation by Dendritic Cells" @default.
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