Phagosomal proteolysis in dendritic cells is modulated by NADPH oxidase in a pH-independent manner
2011; Springer Nature; Volume: 31; Issue: 4 Linguagem: Inglês
10.1038/emboj.2011.440
ISSN1460-2075
AutoresJoanna M. Rybicka, Dale R. Balce, Sibapriya Chaudhuri, Euan R.O. Allan, Robin M. Yates,
Tópico(s)T-cell and B-cell Immunology
ResumoArticle13 December 2011free access Phagosomal proteolysis in dendritic cells is modulated by NADPH oxidase in a pH-independent manner Joanna M Rybicka Joanna M Rybicka Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada Search for more papers by this author Dale R Balce Dale R Balce Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada Search for more papers by this author Sibapriya Chaudhuri Sibapriya Chaudhuri Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada Search for more papers by this author Euan R O Allan Euan R O Allan Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada Search for more papers by this author Robin M Yates Corresponding Author Robin M Yates Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada Search for more papers by this author Joanna M Rybicka Joanna M Rybicka Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada Search for more papers by this author Dale R Balce Dale R Balce Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada Search for more papers by this author Sibapriya Chaudhuri Sibapriya Chaudhuri Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada Search for more papers by this author Euan R O Allan Euan R O Allan Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada Search for more papers by this author Robin M Yates Corresponding Author Robin M Yates Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada Search for more papers by this author Author Information Joanna M Rybicka1,2, Dale R Balce1,2, Sibapriya Chaudhuri1,2, Euan R O Allan1,2 and Robin M Yates 1,2 1Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada 2Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada *Corresponding author. Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, 3330 Hospital Drive NW, HRIC 4AA10, Calgary, Alberta, Canada T2N 4N1. Tel.: +1 403 210 6249; Fax: +1 403 210 8821; E-mail: [email protected] The EMBO Journal (2012)31:932-944https://doi.org/10.1038/emboj.2011.440 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The level of proteolysis within phagosomes of dendritic cells (DCs) is thought to be tightly regulated, as it directly impacts the cell's efficiency to process antigen. Activity of the antimicrobial effector NADPH oxidase (NOX2) has been shown to reduce levels of proteolysis within phagosomes of both macrophages and DCs. However, the proposed mechanisms underlying these observations in these two myeloid cell lineages are dissimilar. Using real-time analysis of lumenal microenvironmental parameters within phagosomes in live bone marrow-derived DCs, we show that the levels of phagosomal proteolysis are diminished in the presence of NOX2 activity, but in contrast to previous reports, the acidification of the phagosome is largely unaffected. As found in macrophages, we show that NOX2 controls phagosomal proteolysis in DCs through redox modulation of local cysteine cathepsins. Aspartic cathepsins were unaffected by redox conditions, indicating that NOX2 skews the relative protease activities in these antigen processing compartments. The ability of DC phagosomes to reduce disulphides was also compromised by NOX2 activity, implicating this oxidase in the control of an additional antigen processing chemistry of DCs. Introduction Although the complexities of dendritic cell (DC) heterogeneity continue to provoke controversy, it is generally accepted that conventional myeloid DCs and macrophages are cell lineages derived from a common precursor, and exist within a functional and phenotypic continuum (Geissmann et al, 2010). Cells that lie on the DC side of this spectrum are more efficient at activating naïve T cells through antigen presentation than those that are skewed towards the macrophage phenotype, which tend to possess greater microbicidal potency (Hume, 2008). Hence, it is reasonable to extrapolate that many cellular processes of macrophages and conventional DCs are similar, or at least share common mechanisms. Since the organization and activity of proteolytic machinery within the phagosomes of DCs are of particular importance to antigen processing, we investigated the control of proteolysis within the phagosome by NADPH oxidase (NOX2) in bone marrow derived-DCs (BMDCs). NOX2 activity has been shown to decrease levels of phagosomal proteolysis in both macrophages and DCs but the proposed mechanisms attributed to this control are dissimilar. Amigorena and co-workers have shown that the efficiency of phagosomal proteolysis in BMDCs is decreased by NOX2 production of reactive oxygen species (ROS) (Savina et al, 2006, 2009; Savina and Amigorena, 2007). Through FACS-based analysis, they presented evidence that NOX2 activity prevents acidification of the phagosome, which, they reasoned, is mediated by superoxide (O2−·) consumption of lumenal protons. Based on these data, they hypothesized that, since lysosomal proteases generally require an acidic pH for optimal activity, the NOX2-induced alkalinization of the phagosomal lumen is responsible for the observed inhibition of phagosomal proteolysis. Similarly, our group has observed a relationship between NOX2 activity and proteolytic efficiency of phagosomes in bone marrow-derived macrophages (BMMØs). However, we demonstrated that this relationship is independent of changes to phagosomal pH (Rybicka et al, 2010). The mechanism through which NOX2 modulates phagosomal proteolysis in these cells was found to be redox-mediated, affecting local cysteine cathepsins through perturbation of the reductive capacity of the phagosome. Since changes to proteolytic efficiency significantly affect antigen processing, and DCs are the dominant antigen-presenting cells, we thought it was prudent to investigate whether NOX2-mediated redox control of phagosomal proteolysis through modification of cysteine cathepsin activity occurs in conventional DCs, as it does in macrophages. Consistent with the findings of Amigorena and colleagues (Savina et al, 2006), we found that NOX2 activity was inversely correlated with phagosomal proteolytic efficiency in BMDCs as well as in the DC-like cell line DC2.4. In contrast to these earlier studies, using highly resolved real-time measurements of phagosomal acidification, we were unable to detect any significant changes in lumenal pH resulting from NOX2 activity. We further demonstrated that NOX2 activity in DCs modifies the redox microenvironment within the phagosomal lumen, leading to compromised disulphide reduction. This is consistent with the oxidative inhibition of phagosomal cysteine cathepsins by NOX2, which could be restored by the addition of exogenous reducing reagents. Collectively, these data support a redox-mediated mechanism of phagosomal protease control by NOX2 in DCs. Results Phagosomal NOX2 activity decreases proteolysis in DCs We first sought to determine the extent and pattern of the NOX2-mediated respiratory burst in BMDCs. These cells were derived from bone marrow of C57Bl/6 mice (WT) using a widely utilized method, and displayed hallmark characteristics of immature DCs (Supplementary Figure S1) (Savina et al, 2006; Geissmann et al, 2010). Using real-time fluorometry, the oxidation of a particle-restricted fluorogenic substrate was measured following coordinated phagocytosis of 3 μm experimental particles. Evidence of the respiratory burst was seen starting at 10 min after internalization and, unlike the pattern observed in macrophages, was sustained beyond 1 h, consistent with prolonged NOX2 association with the phagosome in DCs (Amigorena and Savina, 2010). No detectable substrate oxidation was observed in BMDCs derived from mice deficient in the gp91 subunit of the NOX2 complex (Cybb−/−), or in WT BMDCs treated with the NOX2 inhibitor diphenyleneiodonium (DPI) (Figure 1A and B). To establish whether ROS production by NOX2 in DC phagosomes decreases their proteolytic efficiencies, the hydrolysis of a particle-restricted general protease substrate (DQ-green Bodipy albumin) was measured following its phagocytosis by WT and Cybb−/− BMDCs in the presence or absence of DPI. Consistent with previous findings, the absence of NOX2 activity significantly increased the rate of bulk phagosomal proteolysis (2.38±0.29 fold higher in Cybb−/− BMDCs when compared with untreated WT controls) (Figure 1C and D). The observed differences did not result from differential particle uptake or oxidation of the substrate's fluorophore (Supplementary Figures S2 and S3). Additionally, no differences in protease expression or the proteolytic activity of whole-cell lysates were observed between WT and Cybb−/− BMDCs, indicating that NOX2 specifically impacts phagosomal proteolytic efficiency rather than total cellular proteolytic capacity (Supplementary Figure S4A–C). Figure 1.Generation of ROS by phagosomal NOX2 in BMDCs negatively affects proteolytic efficiency. The generation of ROS (respiratory burst) and general proteolytic activity in phagosomes were evaluated following coordinated phagocytosis of IgG-conjugated 3 μm experimental particles in BMDCs derived from C57Bl/6 (WT) or NOX2-deficient (Cybb−/−) mice. Where indicated, BMDCs were pre-treated with the NOX2 inhibitor DPI (0.5 μM) for 10 min at 37°C prior to phagocytosis. (A, B) Production of ROS in BMDC phagosomes was evaluated by measuring fluorescence released during oxidation of particle-conjugated H2HFF-OxyBURST substrate (λex485 nm; λem520 nm) relative to a calibration fluor Alexa Fluor 594 (λex594 nm; λem620 nm). (C, D) Proteolytic activity was assessed by measuring the amount of fluorescence released during hydrolysis of particle-associated DQ-green Bodipy albumin (λex485 nm; λem520 nm) relative to fluorescence of the calibration fluorophore Alexa Fluor 594 (λex544 nm; λem620 nm). (A, C) Representative real-time traces. (B, D) Average rates of substrate oxidation/hydrolysis over four independent experiments relative to untreated controls. Rates were determined by calculation of the slope of the linear portion of the real-time traces (as described by y=mx+c, where y is the relative fluorescence, m is the slope and x is time) and expressed relative to DMSO-treated WT samples. Error bars represent s.e.m. P-values were calculated using repeated measures one-way ANOVA. Download figure Download PowerPoint NOX2 control of phagosomal proteolysis is locally mediated Control of the delivery of lysosomal constituents to the phagosome through the regulation of the fusion of these compartments is a major regulator of phagosomal physiology (Vieira et al, 2002). To investigate whether NOX2 exerts its inhibitory effect on proteolysis by limiting lysosomal contribution to the phagosome in DCs, we utilized a FRET-based assay that quantifies the accumulation of preformed lysosomal components within the maturing phagosome in real time (Yates et al, 2005; Yates and Russell, 2008). Although we found that the timing and extent of the FRET efficiency between the particle-conjugated phagosomal donor fluor and the lysosomal fluid-phase acceptor fluor was influenced by V-ATPase and calmodulin inhibition (Supplementary Figure S5), no modification of the assay's profile was associated with NOX2 function (Figure 2A and B). This indicates that phagosome–lysosome communication was not influenced by NOX2 activity. To further discount the possibility that NOX2 perturbs phagosomal–lysosomal fusion and thus contributes to differences in phagosomal proteolysis, we followed the phagosomal acquisition of β-galactosidase, a representative lysosomal hydrolase. Consistent with FRET-fusion assay data, we found no significant differences in recruitment of the lysosomal β-galactosidase activity to NOX2-proficient or -compromised phagosomes (Figure 2C and D). Furthermore, phagosomes showed similar relative recruitment of the active forms of cathepsin B, S and L in the presence or absence of NOX2 function, as evidenced by western blot analysis of isolated BMDC phagosomes (Figure 2E and F). These data demonstrate that the decreased proteolytic activity in NOX2-competent phagosomes is not mediated by differential recruitment of lysosomal proteases, indicating that NOX2 affects the activity, rather than the recruitment, of phagosomal proteases. Figure 2.NOX2 activity does not affect phagosome–lysosome communication in BMDCs. (A, B) Lysosomal contribution to the phagosome was measured by evaluating FRET efficiency between a particle-conjugated donor fluor Alexa Fluor 488 (λex485 nm; λem520 nm) and a fluid-phase lysosomal acceptor fluor Alexa Fluor 594 hydrazide (λex485 nm; λem620 nm) relative to the donor fluorescence. RFUs are indicative of the concentration of lysosomal constituents within the phagosome at any given point in time. (C, D) Acquisition of β-galactosidase activity to BMDC phagosomes was measured by following the hydrolysis of the particle restricted fluorogenic β-galactosidase substrate 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (λex485 nm; λem520 nm) relative to a calibration fluor Rhodamine B C10 (λex544 nm; λem620 nm). (A, C) Representative real-time traces. (B, D) Average rates of the acquisition of FRET efficiency or substrate hydrolysis over three independent experiments. Rates were determined by calculation of the slope of the linear portion of the real-time traces (as described by y=mx+c, where y is the relative fluorescence, m is the slope and x is time) and expressed relative to DMSO-treated WT samples. (E, F) Representative western blot images and relative band densities of active forms of cathepsin B (25 kDa), L (28 and 23 kDa) and S (24 kDa) in BMDC phagosomes isolated 1 h following phagocytosis. Volumes of pixels were determined using Quantity One 1-D analysis software and relative densities were calculated relative to DMSO-treated WT BMDC samples over three independent experiments. LAMP-1 was used as a loading control. (B, D, F) Error bars denote s.e.m. No statistical differences between samples were found by ANOVA. Download figure Download PowerPoint Phagosomal NOX2 activity does not significantly affect phagosomal acidification in DCs Utilizing FACS-based measurements of phagosomal pH in BMDCs, Amigorena and colleagues generated data to suggest that NOX2 activity ablated the acidification of phagosomes (Savina et al, 2006). Since phagosomal acidification is largely unaffected by NOX2 activity in BMMØs, we thought it was important to revisit these experiments using real-time acidification assays. These assays dynamically measure the pH of phagosomes in live, undisturbed BMDCs at physiological temperature by single-fluorophore excitation ratio fluorometry. This approach enables measurement of phagosomal pH in a highly resolved, robust and extensively validated manner (Yates and Russell, 2005, 2008; VanderVen et al, 2009). Initially, we utilized the pH-sensitive fluorophore carboxyfluorescein succinimidyl ester (CFSE) covalently bound to IgG-conjugated 3 μm experimental particles in a population-based format. Consistent with the findings in BMMØs, we found no statistical difference between the final pH of phagosomes with or without NOX2 function in BMDCs (Figure 3A and B). Similar results were generated using non-opsonized experimental particles targeted to the mannose receptor (Supplementary Figure S6) and pH measurements performed using an alternative pH-sensitive fluorophore Oregon Green succinimidyl ester (OGSE) (Supplementary Figure S7). In the original series of experiments, however, there was a statistically insignificant trend towards slightly more acidified phagosomes (0.17±0.056 units) in Cybb−/− BMDCs (Figure 3B). To calculate the theoretical impact of this small difference in pH on phagosomal proteolysis, phagosomal pH measurements were regressed against a curve describing the proteolytic efficiency of BMDC lysosomal contents at different pH values (Figure 3C). It was found that, even if significant, the small NOX2-mediated alkalinization would theoretically increase, not decrease, the proteolytic efficiency of the phagosome of BMDCs by ∼8%. Indeed, the phagosomal pH would have to increase to above a value of 6.0 before the total proteolytic efficiency would be adversely affected (Figure 3C; Supplementary Figure S8). Presumably, in this scenario, lysosome fusion with these largely unacidified phagosomes would also be compromised, which was not evidenced by phagosome–lysosome fusion profiles (Figure 2A and B) (Supplementary Figure S5) (Clague et al, 1994; van Weert et al, 1995; Yates et al, 2005). Figure 3.NOX2 activity does not significantly compromise phagosomal acidification in BMDCs. Phagosomal pH was measured by excitation ratio fluorometry using the pH-sensitive fluor CFSE (λex1485 nm, λex2450 nm; λem520 nm) conjugated to IgG-coupled experimental particles followed by ratio regression to a standard curve. (A) Representative real-time acidification profile in the phagosomes of WT and Cybb−/− BMDCs in the presence or absence of 0.5 μM DPI. (B) Average phagosomal pH at 45 min following phagocytosis from four independent experiments. (C) Curve describing the effect of pH on the proteolytic efficiency of total lysosomal extract from BMDCs. The curve was generated by measuring proteolytic efficiency of magnetically isolated lysosomal extract of BMDCs using the fluorogenic substrate DQ-green Bodipy albumin (λex485 nm; λem520 nm) in buffers of known pH; n=3. (D) Representative pseudo-colour ratio images (λex1488 nm; λem520 nm/λex2458 nm; λem520 nm) of BMDCs at time 0 and 60 min. Ratio/overlay images were generated using Leica Application Suite Advanced Fluorescence software. (E) Average real-time phagosomal acidification profile of WT and Cybb−/− BMDCs measured using confocal microscopy in the presence or absence of 0.5 μM DPI or 100 nM concanamycin A (ConA) (V-ATPase inhibitor) from three independent experiments. Extracellular beads were included as an additional control (beads). Error bars denote s.e.m. (F) Final pH of individual phagosomes from three independent experiments. Download figure Download PowerPoint Since the methods of BMDC differentiation could potentially explain phenotypic differences between the current study and that of Savina et al (2006), we repeated these experiments in alternative, widely used, conventional DC types. Consistent with our earlier findings, NOX2 activity negatively impacted phagosomal proteolysis, but not acidification, in the immortalized DC line DC2.4 (Shen et al, 1997) and BMDCs derived using standard protocols with recombinant GM-CSF (Supplementary Figures 9A–F and S10A–G). As fluorophore peroxidation and chlorination has previously led to inaccurate pH measurement in the presence of oxidative radicals and their products (Segal et al, 1981; Hurst et al, 1984), we tested the fluorescent stability of both CFSE and OGSE within BMDC phagosomes. Firstly, CFSE-generated acidification profiles were not perturbed in the presence of the peroxidase inhibitor sodium azide (Supplementary Figure S11) (Jankowski et al, 2002). Secondly, CFSE- and OGSE-coupled experimental particles displayed identical pH-sensitive fluorescent properties following recovery from either WT or Cybb−/− BMDC phagosomes (Supplementary Figure S12A and B). Together, these data indicate that the fluorescent properties of the pH-sensitive fluorophores used in this study were not affected by NOX2 products generated in BMDC phagosomes. It is interesting to note however, that in contrast to CFSE and OGSE, the fluorescent stability of pH-sensitive fluorophores pHrodo (Invitrogen) and cypHer (GE Healthcare) were found to be significantly affected by NOX2 activity. These two newer-generation fluorophores showed vastly disparate fluorescent properties following recovery from WT and Cybb−/− BMDC phagosomes, suggesting that they are inappropriate for quantitative measurement of phagosomal pH in the presence of ROS (Supplementary Figure S12C and D). In addition to the measurement of lumenal pH across populations of synchronized BMDC phagosomes, we measured the pH of individual BMDC phagosomes containing CFSE-coupled, IgG-opsonized experimental particles under physiological conditions using real-time fluorometric confocal microscopy (Figure 3D and F). Consistent with previous data, we found that NOX2 activity did not influence the rate or extent of the acidification of individual BMDC phagosomes, and little heterogeneity of pH between phagosomes was observed (Figure 3F). To further test the effect of NOX2 on the heterogeneity of phagosomal acidification in BMDCs, we measured the accumulation of the acidotropic probe LysoTracker® Green (LTG) in mature phagosomes. LTG is an acid-selective organellar probe, which accumulates in acidified, but not unacidified, lysosomes and phagosomes. Consistent with previous results, mature (90-min) phagosomes containing IgG-opsonized or mannosylated silica experimental particles accumulated similar amounts of pulsed LTG in WT or Cybb−/− BMDCs (Supplementary Figure S13). Similar observations were made in phagosomes containing 3 μm latex beads used by Savina et al (2006). Interestingly, WT BMDC phagosomes containing zymosan displayed increased heterogeneity of phagosomal pH, with a greater proportion of unacidified phagosomes when compared with Cybb−/− BMDCs. The mechanism underlying the observed heterogeneity in this particular case is undetermined. Nevertheless, the vast majority of phagosomes containing zymosan, latex, IgG-coupled and mannose-coupled experimental particles acidified in NOX2-competent BMDCs. Phagosomal NOX2 activity is dependent on charge compensation provided by translocation of protons into the phagosome To further explore the relationship between NOX2 activity and phagosomal acidification in BMDCs from a stoichiometric perspective, we investigated the dependence of NOX2 activity on the translocation of protons into the phagosomal lumen by vacuolar ATPase (V-ATPase) and through the voltage-gated proton channel 1 (Hv1). Since NOX2 translocates electrons from cytosolic NADPH to O2 within the phagosome, compensatory movement of counter-ions across the phagosomal membrane is required to sustain NOX2 activity (Henderson et al, 1987; DeCoursey et al, 2003). The movement of protons across the plasma membrane provide the majority of charge compensation during the extracellular respiratory burst in neutrophils (Gabig et al, 1984; Takanaka and O'Brien, 1988), and the same mechanism has been proposed to support the phagosomal NOX2 activity in these cells (Morgan et al, 2009; DeCoursey, 2010). Should this be the case in DC phagosomes, then proton scavenging by superoxide and the proton counter-ion influx needed to sustain superoxide generation would occur at a 1:1 ratio and thus, theoretically, not influence lumenal pH. We first investigated the relationship between NOX2 and V-ATPase in BMDC phagosomes. If NOX2 activity is independent of charge compensation provided by V-ATPase activity, NOX2 would still function after inhibition of V-ATPase. In this scenario, there would be no active acidification of the phagosome but unimpeded superoxide production, which would result in the rapid depletion of existing lumenal protons and dramatic alkalinization of the phagosome. To test this, we used the specific V-ATPase inhibitor concanamycin A to temporally inhibit active phagosomal acidification in BMDCs in the presence or absence of the NOX2 inhibitor DPI. When the V-ATPase complex was inhibited before phagocytosis, NOX2-competent phagosomes only displayed marginal lumenal alkalinization at this neutral pH (0.30±0.090 increase in pH) (Figure 4A). When the V-ATPase was inhibited in fully acidified phagosomes, NOX2 had no observable influence on the rates of the subsequent alkalinization of the phagosome, which could be entirely attributed to passive proton leak (Lukacs et al, 1991). To further investigate the relationship of V-ATPase and NOX2 activity, we evaluated the magnitude of the respiratory burst in the presence of concanamycin A by following the oxidation of a particle-restricted fluorogenic substrate. We found that inhibition of V-ATPases profoundly decreased the NOX2-associated substrate oxidation within the phagosome (Figure 4B and C). Although the rates of substrate oxidation are possibly affected by pH directly, these findings, in conjunction with the acidification data, strongly suggest that NOX2 activity is compromised in the absence of the compensating electrogenic activity provided by phagosomal V-ATPase. In addition to V-ATPase, another potential source of charge compensation for NOX2 activity is the voltage-gated proton channel Hv1 (Ramsey et al, 2006). Selective movement of protons across the plasma and phagosomal membranes through this divalent cation (Zn2+, Cd2+)-sensitive proton channel has been proposed to provide the majority of charge compensation during NOX2 activity in neutrophils (Morgan et al, 2009; Ramsey et al, 2009; DeCoursey, 2010). We thus investigated the possible role of Hv1 during the phagosomal respiratory burst in BMDCs. Both WT and Cybb−/− BMDCs expressed similar amounts of Hv1 mRNA as determined by RT–PCR and quantitative PCR (Supplementary Figure S14). BMDCs derived from WT and Hv1−/− mice showed comparable rates and extents of phagosomal acidification in the presence and absence of NOX2 activity (Figure 4D and E). The respiratory burst, however, was found to be significantly compromised in Hv1−/− phagosomes or in the presence of the Hv1 inhibitor ZnCl2 (31.18%±5.26 and 44.47%±9.44 reduction, respectively) and completely compromised in the absence of Hv1 and V-ATPase activity (Figure 4F and G). These data suggest that a major proportion of phagosomal NOX2 activity in BMDCs is dependent on the charge compensation provided by proton translocation into the phagosomal lumen by V-ATPase and Hv1. Overall, these findings are consistent with maintained phagosomal acidification during NOX2 activity, as evidenced by direct measurement of pH (Figure 3), and support the existence of a pH-independent control mechanism of phagosomal proteolysis in DCs. Figure 4.Dependence of NOX2 activity on charge compensation provided by V-ATPase and Hv1 in BMDC phagosomes. (A) Representative real-time acidification profiles of phagosomes containing IgG-, CFSE-coupled experimental particles in WT and Cybb−/− BMDCs (± 0.5 μM DPI) with the addition of the V-ATPase inhibitor concanamycin A (100 nM) (ConA) prior to, or after, acidification of the phagosome. (B, C, F, G) NOX2 activity in BMDC phagosomes was evaluated by measuring fluorescence released during oxidation of particle-conjugated H2HFF-OxyBURST substrate (λex485 nm; λem520 nm) relative to a calibration fluor Alexa Fluor 594 (λex594 nm; λem620 nm) in the presence of V-ATPase inhibitor concanamycin A (100 nM) (ConA), Hv1 inhibitor ZnCl2 (50 μM) and/or DPI (0.5 μM DPI). (B, F) Representative real-time traces. (C, G) Average rates of substrate oxidation over three independent experiments relative to untreated controls. Error bars represent s.e.m. P-values were calculated using ANOVA. (D, E) Acidification of phagosomes containing IgG-, CFSE-coupled experimental particles in WT and Hv1−/− BMDCs (±0.5 μM DPI, ±100 nM ConA). (D) Representative real-time acidification profiles. (E) Average of final phagosomal
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