NADPH oxidase modifies patterns of MHC class II-restricted epitopic repertoires through redox control of antigen processing.

NADPH Oxidase Modifies Patterns of MHC Class II−Restricted Epitopic Repertoires through Redox Control of Antigen Processing Euan R. O. Allan, Pankaj Tailor, Dale R. Balce, Payman Pirzadeh, Neil T. McKenna, Bernard Renaux, Amy L. Warren, Frank R. Jirik and Robin M. Yates J Immunol 2014; 192:4989-5001; Prepublished online 28 April 2014; doi: 10.4049/jimmunol.1302896 http://www.jimmunol.org/content/192/11/4989

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The Journal of Immunology

NADPH Oxidase Modifies Patterns of MHC Class II–Restricted Epitopic Repertoires through Redox Control of Antigen Processing

The chemistries within phagosomes of APCs mediate microbial destruction as well as generate peptides for presentation on MHC class II. The antimicrobial effector NADPH oxidase (NOX2), which generates superoxide within maturing phagosomes, has also been shown to regulate activities of cysteine cathepsins through modulation of the lumenal redox potential. Using real-time analyses of lumenal microenvironmental parameters, in conjunction with hydrolysis pattern assessment of phagocytosed proteins, we demonstrated that NOX2 activity not only affects levels of phagosomal proteolysis as previously shown, but also the pattern of proteolytic digestion. Additionally, it was found that NOX2 deficiency adversely affected the ability of bone marrow–derived macrophages, but not dendritic cells, to process and present the I-Ab–immunodominant peptide of the autoantigen myelin oligodendrocyte glycoprotein (MOG). Computational and experimental analyses indicated that the I-Ab binding region of the immunodominant peptide of MOG is susceptible to cleavage by the NOX2-controlled cysteine cathepsins L and S in a redox-dependent manner. Consistent with these findings, I-Ab mice that were deficient in the p47phox or gp91phox subunits of NOX2 were partially protected from MOGinduced experimental autoimmune encephalomyelitis and displayed compromised reactivation of MOG-specific CD4+ T cells in the CNS, despite eliciting a normal primary CD4+ T cell response to the inoculated MOG Ag. Taken together, this study demonstrates that the redox microenvironment within the phagosomes of APCs is a determinant in MHC class II repertoire production in a cell-specific and Ag-specific manner, which can ultimately impact susceptibility to CD4+ T cell–driven autoimmune disease processes. The Journal of Immunology, 2014, 192: 4989–5001.

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hagocytosed or endocytosed exogenous Ags must be proteolytically processed within the phagosomes and endosomes of the APCs to be complexed with MHC class II (MHC-II) and presented to CD4+ T cells. Although intralumenal proteolysis is a prerequisite for generating oligopeptides of 15– 24 aa in length for MHC-II, too much proteolysis is thought to be detrimental to Ag processing efficiencies as it can over-digest the *Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada; † Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada; ‡Department of Chemistry, Faculty of Science, University of Calgary, Calgary, Alberta T2N 1N4, Canada; and x Department of Ecosystem and Public Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada Received for publication October 28, 2013. Accepted for publication March 28, 2014. This work was supported by the Canadian Institutes of Health Research and the Multiple Sclerosis Society of Canada. Graduate student support was provided by the endMS training network and Alberta Innovates: Health Solutions. Address correspondence and reprint requests to Robin M. Yates, Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, 3330 Hospital Drive NW, HRIC 4AA10, Calgary, AB T2N 4N1, Canada. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: BMMf, bone marrow–derived macrophage; BMDC, bone marrow–derived dendritic cell; DC, dendritic cell; EAE, experimental autoimmune encephalomyelitis; eGFP, enhanced GFP; Em, emission; Ex, excitation; FSC, forward scatter; HEL, hen egg white lysozyme; MHC-II, MHC class II; MOG, myelin oligodendrocyte glycoprotein; NOX2, NADPH oxidase; PDB, Protein Data Bank; qPCR, quantitative real-time PCR; RFU, relative fluorescent unit; rMOG, recombinant MOG; ROS, reactive oxygen species; SSC, side scatter; TFA, trifluoroacetic acid. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302896

oligopeptides (1). It has thus become increasingly apparent that tight control over the level of proteolysis within these compartments is necessary to generate and preserve the antigenic oligopeptides required for productive Ag presentation to T cells (2, 3). However, beyond the overarching correlation of levels of proteolysis and processing efficiency, the intricacies of the effect of relative protease activities upon the pattern of Ag microdissection remain less evident. Theoretically, because antigenic proteolysis is performed by numerous and diverse proteases (each with differing substrate and cleavage-site preferences), the relative local abundance and activity of each protease could affect the pattern of proteolysis and hence the relative availability of different peptides for loading onto MHC-II. To complicate this paradigm, identical CD4+ T cell epitopes must be generated by different APCs (notably thymic epithelial cells, dendritic cells [DCs], B cells, and macrophages) in different tissue environments and during different inflammatory states in order for effector CD4+ T cells to function. Whether this perceived complexity of MHC-II epitope generation translates into modification of T cell immunity during an immune response, or whether relative epitopic abundance within Ag processing compartments exceeds saturable levels of MHC-II (such that subtle modification of processing patterns would be of no consequence) is largely undetermined. It was recently discovered that the antimicrobial effector NADPH oxidase (NOX2) negatively regulates the levels of proteolysis within the maturing phagosome of macrophages (4, 5) and DCs (3, 6, 7). NOX2, a multiprotein complex expressed in phagocytes, is rapidly assembled on the early phagosomal membrane where it oxidizes cytosolic NADPH to convert molecular oxygen to superoxide within the phagosomal lumen. Because


Euan R. O. Allan,*,† Pankaj Tailor,*,† Dale R. Balce,*,† Payman Pirzadeh,*,†,‡ Neil T. McKenna,*,† Bernard Renaux,*,† Amy L. Warren,x Frank R. Jirik,† and Robin M. Yates*,†

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Materials and Methods Mice and cells C57BL/6 (wild-type [WT]) mice and the congenic mouse strains B6.129SCybbtm1Din/J (Cybb2/2), B6 (Cg)-Ncf1m1J/J (Ncf), B6.Cg-Tg(TcraTcrb) 425Cbn/J (OTII), and C57BL/6-Tg(Tcra2D2,Tcrb2D2)1Kuch/J (2D2) were purchased from The Jackson Laboratory and bred in-house under identical husbandry. Cybb2/2 mice lack the gp91phox (catalytic) subunit of NOX2, whereas Ncf mice lack the p47phox (activating) subunit of NOX2. OTII mice have a transgenic CD4+ TCR specific for OVA323–339 in the context of I-Ab, and 2D2 mice express a transgenic CD4+ TCR (Vb11 TCR/Va3.2 TCR) specific for MOG35–55 in the context of I-Ab. All animal research was performed according to protocols approved by the University of Calgary Animal Care and Use Committee and in accordance with the Canadian Council of Animal Care. Bone marrow–derived macrophages (BMMfs) and bone marrow–derived DCs (BMDCs) were derived from 8to 12-wk-old male mice using L929-conditioned media or conditioned media derived from the supernatant of Ag8653 melanoma cells transfected

with murine GM-CSF cDNA, respectively, as previously described (3, 5, 16, 17). BMMfs were activated overnight (18 h) with rIFN-g (100 U/ml, PeproTech) for Ag presentation assays (5). Female mice used for EAE studies were immunized between 8 and 10 wk and age-matched within experiments (10, 18, 19).

Protein and peptide synthesis MOG35–55 was synthesized at the University of Calgary (University of Calgary’s Peptide Services, Calgary, AB, Canada). Recombinant protein corresponding to the extracellular domain of MOG1–125 (rMOG) was cloned with a carboxyl terminal 6xHis tag, expressed using Rosetta Blue (DE3) Escherichia coli and purified using Ni-NTA agarose beads (Qiagen). Endotoxin was removed to ,0.005 endotoxin unit/mg (E-Toxate kit, Sigma-Aldrich) using Endotoxin Affisorbant agarose (polymyxin B Separopore, bioWORLD, Dublin, OH), according to the manufacturers’ directions.

Flow cytometry Flow cytometry data were acquired using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ) and analyzed using FlowJo software v8.6 (Tree Star, Ashland, OR). Unless otherwise indicated, leukocyte populations were selected using forward scatter/side scatter (FSC/SSC) and samples were measured with a minimum of 2.5 3 104 counts. Unless otherwise noted, Abs were purchased from BD Biosciences.

Fluorometric phagosomal analysis Three-micrometer IgG-conjugated silica experimental particles were used to evaluate intraphagosomal reactive oxygen species (ROS) generation, proteolysis and cysteine cathepsin activity, intraphagosomal pH, and disulfide reduction in populations of live BMMfs that were prepared as previously described (4, 5, 20–22). Quantification was performed using a Safire microplate reader (Tecan, Ma¨nnedorf, Switzerland), a FLUOstar Optima fluorescent plate reader (BMG Labtech, Ortenberg, Germany), or an EnVision multilabel reader (PerkinElmer, Waltham, MA) at 37˚C, at a multiplicity of infection of two to three particles per cell, in an assay buffer containing PBS supplemented with 1 mM calcium chloride, 2.7 mM potassium chloride, 0.5 mM magnesium chloride, 5 mM dextrose, and 0.25% gelatin (4, 23). Real-time traces are shown in relative fluorescent units (RFU), which were calculated by dividing the substrate fluorescence (SFRT) by the average calibration fluorescence (CF) for a particular time point (RFU = SFRT/CF) (7). The average rates (taken from 30 to 80 min after phagocytosis) of oxidation, proteolysis, and cathepsin activities were determined by the linear portions of the real-time traces and made relative to the internal controls as indicated in each figure (y = mx + c, where y indicates relative fluorescence, m indicates gradient, and x indicates time). Intraphagosomal ROS generation by NOX2 in APCs was assessed via the quantification of particle-restricted H2HFF-OxyBurst substrate (Molecular Probes) relative to Alexa Fluor 594–succinimidyl ester (calibration fluorescence) as previously described (4). The intraphagosomal total protease activity and the intraphagosomal hydrolytic activity of cathepsin B/S/L in APCs were assessed by recording the rate of substrate-liberated fluorescence relative to the calibration fluorescence using the particle-bound fluorogenic DQ green Bodipy albumin (DQ-albumin) (Molecular Probes) and (biotin-LC-Phe-Arg)2-rhodamine 110 (provided by David Russell, Cornell University, Ithaca, NY), respectively (4, 5, 21, 23, 24). Intraphagosomal pH was measured by monitoring the excitation ratio of particle-restricted CFSE (excitation [Ex]/emission [Em] of 490/520 and 450/520 nm), followed by regression to a third-order polynomial standard curve (generated using experimental particles in buffers with known pH) as previously described (4, 5, 21, 24). Intraphagosomal reductase activity was examined using the rate of fluorescence liberated from Bodipy FL Lcystine (Molecular Probes), a self-quenched cystine-based fluorogenic substrate conjugated to dextran-coated experimental particles, relative to Alexa Fluor 594 succinimidyl ester (calibration fluorescence, Molecular Probes) as previously described (4, 5). Phagocytic index was determined using 3-mm IgG-opsonized, BSA-coated silica experimental particles labeled with Alexa Fluor 594 succinimidyl ester given to BMMf monolayers. Trypan blue (EMD Chemicals) (0.01% in PBS) was used to quench the Alexa Fluor 594 fluorescence of extracellular particles. The phagocytosed particles in three separate images from each well were counted using the 403 (numerical aperture, 0.75) objective on an Olympus IX70 fluorescence microscope (Olympus, Center Valley, PA) (4). Rates of endocytosis/pinocytosis of Alexa Fluor 488–labeled 70-kDa dextran were measured in BMMfs. In brief, APCs were pulsed with medium containing 500 mg/ml Alexa Fluor 488–labeled dextran for 18 h, washed, and chased in assay buffer for 4 h. Fluorescence was measured using a FLUOstar Optima fluorescent plate reader. Background was deducted and values


NOX2 activity dramatically oxidizes the otherwise reductive microenvironment of the phagosomal lumen, the local cysteine cathepsins (which require a reductive environment for activity), but not the non–cysteine proteases, such as the aspartic cathepsins, are temporarily inactivated (4, 5, 7). Over and above the impact on general proteolytic efficiency, we predicted that the impact of NOX2 on the activities of certain protease subsets within maturing phagosomes and endosomes would affect the pattern of proteolytic digestion and thus the relative probabilities of specific oligopeptides being generated. NOX2 activity also inhibits the reduction of disulfide bonds within the phagosome (5). This could limit the intraphagosomal denaturation of Ags that contain disulfide bonds and potentially hide vast stretches of an Ag’s polypeptide sequence from proteolysis. It may also limit the presentation of specific oligopeptides that contain a disulfide linkage in the native protein (8, 9). Taken together, we hypothesized that NOX2’s control over proteolysis and disulfide reduction would affect not only the efficiency of Ag processing, but also the repertoire of antigenic peptides available for MHC-II presentation. Murine experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of multiple sclerosis, is primarily driven by myelin Ag-specific autoreactive CD4+ T lymphocytes (10). CD4+ T cells must first be activated and expanded in the periphery (primarily by DCs in secondary lymphoid organs) following immunization with myelin-derived proteins/peptides (e.g., myelin oligodendrocyte glycoprotein [MOG]). Reactivation of these expanded effector CD4+ T cells by APCs within the subarachnoid space triggers a local inflammatory response leading to oligodendrocyte damage and death (11, 12). Further phagocytosis of myelin debris by APCs (particularly macrophages) increases the presentation of myelin-derived Ags to effector T cells, creating a positive feedback loop that amplifies the inflammatory process to a level that is clinically apparent. Hence, the local and recruited macrophages act as the central “gatekeepers” of T cell reactivation and ultimately the initiation and propagation of clinical EAE (11, 13–15). Thus, EAE presents a good model for studying the generation of MHC-II–restricted autoepitopes, from both exogenous and endogenous sources, by different APCs in different tissues. In the present study, to our knowledge we demonstrate for the first time that the redox environment within phagolysosomes differentially influences local protease activities resulting in 1) altered patterns of antigenic processing, 2) modified activation of specific CD4+ T cell clones, and 3) altered susceptibility to autoimmune clinical disease perpetrated by a cysteine/cathepsin– susceptible immunodominant peptide epitope. Whereas most therapies for autoimmune disorders target T cell pathways, these findings suggest that autoreactive T cell responses may also be attenuated through manipulation of Ag processing chemistries.

NOX2 MODULATES PATTERNS OF Ag PROCESSING

The Journal of Immunology expressed relative to WT for each experiment (n = 3). Representative images were taken using the 403 (numerical aperture, 0.75) objective on an Olympus IX70 fluorescence microscope.

Assessment of pattern of intraphagosomal proteolysis

In vitro assessment of Ag processing and presentation To evaluate specific changes to presented antigenic peptide repertoires from phagocytosed Ag processed in the presence or absence of an NOX2, four I-Ab–restricted CD4+ T cell hybridomas lines that each responds to different HEL epitopes (Hb1.9 [HEL20–35], H30.44 [HEL31–47], H46.13 [HEL48–62], and B04 [HEL74–90]) (provided by Dr. Lars Karlsson, Johnson & Johnson, San Diego, CA) (25, 26) were stably transfected with a pcDNA3 construct that contained enhanced GFP (eGFP) under the control of a NFATenhanced IL-2 promoter (pNFATeGFP) as previously described (25–28). Hence, upon productive Ag presentation with the hybridoma’s cognate I-Ab– restricted Ag, the hybridoma would express eGFP. BMMfs from WT and Cybb2/2 mice were exposed to 1 mg/ml HEL for 6 h and individual hybridomas were added and incubated with the BMMfs for 16 h. Presentation efficiency of each HEL epitope was determined by measuring eGFP expression by flow cytometry and is presented relative to GFP expression induced by nonspecific hybridoma activation by 50 ng/ml PMA and 1 mg/ml ionomycin (25, 26). Ag presentation efficiencies were also assessed using OVA and MOG as Ags utilizing the TCR transgenic mouse models OTII and 2D2, respectively. BMMfs and BMDCs from WT, Cybb2/2, and Ncf mice were exposed to OVA323–339 (10, 25, and 50 mg/ml), OVA (10, 25, and 50 mg/ml), MOG35–55 (25 mg/ml), rMOG (10 and 25 mg/ml), myelin sonicates (purified from mouse brain homogenate as previously described; 25 and 50 mg/ml), and 3mm silica experimental particles with MOG1–125 adsorbed in the presence or absence of IgG opsonization (2.5 mg/ml, three to five beads per APC) (4, 5, 29) for a period of 6 h (30–35). APCs were washed extensively, and naive OTII or 2D2 splenocytes were added and incubated with the APCs for 16 h. Presentation efficiencies of OVA323–339 or MOG35–55 were determined by measuring surface expression of the early activation marker (CD69) and IL2Ra (CD25) on CD4+ OTII/2D2 T cells (gated by FSC/SSC, CD4+) via flow cytometry (35). Similar to previous reports, pilot experiments revealed that a range of pulsed Ag concentration between 10 and 50 mg/ml gave the greatest dynamic T cell response in our experimental setup and was used to determine Ag concentrations used in all presentation experiments (Supplemental Fig. 2A–E) (30–35).

Induction of EAE EAE was induced using standard protocols as described (10, 18, 36). In brief, 8- to 10-wk-old female WT, Cybb2/2, and Ncf mice were anesthetized with ketamine-xylazine and injected s.c. with an emulsion of 50 mg MOG35–55 (24 of 25 WT, 11 of 37 NOX2-deficient), 200 mg MOG71–90, 200 mg MOG101–120, or 200 mg rMOG in CFA (0.5 mg/ml Mycobacterium butyricum in paraffin oil) (BD Difco, Franklin Lakes NJ) in a total volume of 200 ml split between each flank. Pertussis toxin (300 ng) (List Biological Laboratories, Campbell, CA) was injected i.p. (pH 7, in saline) on day 0 and day 2. MOG71–90 and MOG101–120 were not sufficient to induce EAE in any genotype. Mice were weighed and clinically scored each day for 40 d. In brief, scores were: 0, asymptomatic; 0.5, tail weakness; 1, limp tail; 1.5, hindlimb limping; 2, hindlimb weakness; 2.5, partial hindlimb paralysis; 3, complete hindlimb paralysis; 3.5, hindlimb paralysis with forelimb weakness; 4, forelimb paralysis; 4.5–5, morbidity/death (10). Mice that reached a clinical score of 4 were euthanized. Control mice injected with saline/CFA/pertussis toxin did not develop clinical symptoms of EAE. Where indicated, mice were sacrificed 12 d after injection, or at the peak of the clinical score to collect tissues (brain, spinal cord, inguinal lymph nodes, spleen, and serum) for analysis by histopathology, quanti-

tative real-time PCR (qPCR), Luminex, or flow cytometry (10, 37). To specifically measure efficiencies of reactivation of MOG35–55-specific CD4+ T cells by APCs within the CNS, CD4+ T cells from 2D2 mice were expanded and adoptively transferred into preclinical EAE-induced WT and Cybb2/2 mice. In brief, splenocytes were harvested from 2D2 mice and cultured in T cell medium (RPMI 1640 with 10% FCS, 10 mM 2-ME) supplemented with 0.5 ng/ml IL-12 (R&D Systems) and 20 mg/ml MOG35–55 for 72 h as previously described (38). Following a 24-h resting period in T cell media without IL-12 and MOG35–55, 2D2 cells were adoptively transferred (5 3 106 cells/mouse in 100 ml PBS) into EAEinduced mice via i.p. injection on day 7 after induction. MOG-specific 2D2 CD4+ cell recruitment to, and reactivation within, the CNS were determined on day 10 after induction, following isolation and flow cytometric analysis of lumbar spinal cord tissue.

Flow cytometry of CNS tissue Spinal cord-infiltrating and resident leukocytes were isolated from EAE mouse spinal cords at 12 d after induction, at the clinical peak of disease, or preclinical symptoms at 10 d for the harvest of adoptively transferred 2D2 CD4+ T cells, via a discontinuous Percoll gradient as previously described (19). Cells were immunostained for CD4 (PerCP), CD8 (PE), CD3 (FITC), CD11b (PE), CD45 (PE, FITC), B220 (FITC), and CD11c (FITC) in the following combinations—CD4/8/3 (T cells), CD11b/45 (macrophages/ microglia), B220/CD45 (B cells), and CD11b/CD11c (macrophages/ DCs)—and analyzed by flow cytometry. Leukocytes were selected by FSC/ SSC, and specific populations were identified using Abs against lineagespecific markers where indicated. Absolute numbers of each cell type were calculated from the percentages after gating: absolute number of cells from leukocyte isolation 3 proportion of leukocytes (FSC/SSC) 3 proportion of positively stained cells. CD4 and CD8 cells were identified based on CD4 and CD8 staining on the CD3+ lymphocyte population. Macrophage and microglia were differentiated by high (macrophage) or low (microglia) CD45 expression. CD4+ T cells were stained for intracellular Foxp3, IFN-g, and IL-17, and absolute cell numbers were calculated as described above. Additionally, the absolute number of infiltrating spinal cord MOG35–55/I-Ab-positive CD4+ T cells after 12 d of EAE with MOG35–55 was examined. Spinal cord cells were isolated (as described above) and were incubated with MOG35–55/I-Ab (PE) or control tetramers (CLIP/I-Ab; PE) (National Institutes of Health Core Facility, Emory University, Atlanta, GA) at 37˚C overnight, washed, and stained with anti-CD4 (PerCP). CD4+ cells were gated on MOG35–55/I-Ab tetramer or control CLIP tetramer. Leukocytes were selected by FSC/SSC, and absolute numbers of tetramer+ cells were calculated from percentages after gating: absolute number of cells from leukocyte isolation 3 proportion of leukocytes (FSC/SSC) 3 proportion of positively stained CD4 cells 3 proportion of MOG35–55/I-Ab tetramer+ cells. Finally, spinal cord cells were isolated (as described above) from adoptive transfer mice 10 d after EAE induction/3 d after adoptive transfer of 2D2 splenocytes. Leukocytes were selected based on FSC/SSC, and from this population 2D2 CD4+ T cells were identified based on CD4/Vb11 TCR/Va3.2 TCR expression. The activation status of these T cells was assessed by additional identification of CD25 and intracellular IFN-g+ populations.

T lymphocyte proliferation ex vivo Lymph node cells were isolated from inguinal lymph nodes of mice 12 d after inoculation with rMOG/CFA and cultured in a 96-well U-bottom plates at a density of 2.5 3 105 cells/well. Cells were restimulated with 25 mg/ml of MOG35–55, MOG71–90, MOG101–120, or rMOG for 48 h at 37˚C before being pulsed with 1 mCi [3H]thymidine (MP Biomedicals, Irvine, CA) for 18 h at 37˚C. Cells were harvested and the incorporation of radioactive thymidine was measured by scintillation (1450 MicroBeta TriLux; PerkinElmer). Stimulation index is the proliferative response relative to the maximum proliferation induced by the positive control, rMOG, after no peptide was removed as background.

Histopathology CNS lesion location, inflammation, and extent of demyelination were assessed by histopathological examination of CNS tissues from MOG35–55 and rMOG immunized mice sacrificed at day 12 or at peak clinical score (36, 39). Formalin-fixed cross-sections and longitudinal sections of brain, cerebellum, and cervical, thoracic, and lumbar spinal cord were stained with Luxol fast blue and H&E (Goodman Cancer Centre Histology Core Facility, Montreal, QC, Canada) and examined by a boarded veterinary pathologist in a blinded fashion. Histopathological assessment of inflammation and demyelination was quantified according to Racke et al. (40). In brief, inflammation scores were: 0, no inflammatory cells; 1, few scattered inflammatory cells; 2, organization of inflammatory infiltrates into peri-


BMMfs derived from WT and Cybb2/2 mice were exposed to IFN-g (100 U/ml, 18 h) and given 90 min to phagocytose/process Alexa Fluor 488–hen egg white lysozyme (HEL) (Alexa Fluor 488, Invitrogen; HEL, SigmaAldrich) covalently coupled to IgG-opsonized 3-mm silica experimental particles (multiplicity of infection of three to four beads per BMMf). BMMfs were lysed in 0.1% trifluoroacetic acid (TFA) in the presence of protease inhibitor mixture 1 (Calbiochem/Merck, Darmstadt, Germany) and passed through a centrifugal 10-kDa microconcentrator (Nanosep 10K Omega, Pall Canada, St. Laurent, QC, Canada). Peptides in the filtrate were separated by reverse-phase HPLC using a Supelco Ascentis Express C18 column (10 cm length, 2.7 mm pore) (Sigma-Aldrich) using a 0–30% acetonitrile gradient with 0.1% TFA. Fluorescently labeled peptides were detected with a Waters 2475 fluorescent detector (lEx of 488 nm/lEm of 520 nm). Relative peptide abundance was determined using area under the curve for relevant peaks and made proportional to WT samples.

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NOX2 MODULATES PATTERNS OF Ag PROCESSING 1024) is plotted for each binding region within the first 125 aa of MOG (rMOG). The software algorithm SitePrediction (49) was used to predict the cleavage patterns of MOG35–55 by cysteine cathepsins using input sites from the MEROPS database. The illustration (see Fig. 7) was drafted by Bill Zaun of ZaunArt (Ft. Collins, CO). The MOG35–55/I-Ab complex (see Fig. 7) is adapted from Carrillo-Vico et al. (50).

Structural analysis of cathepsin/MOG complexes

qPCR and qPCR array

The crystal structures of cathepsins L and S bound to ligands were obtained from the Protein Data Bank (PDB) database, 3IV2 (41) and 3OVX (42), respectively. The ligand molecules were eliminated and the engineered residues were mutated back to WT residues. Missing residues from the structures were added through structural alignments with other available PDB files. The 1PY9 crystal structure was used for MOG (43). Similar to cathepsins, missing residues were added to the structure by comparing to other available structures on the PDB database. For performing the docking process, the ZDOCK server was used (44, 45). The PDB files of cathepsins (L and S) and MOG were uploaded as receptors and ligands, respectively. Other than the active site residues and those comprising the loops around the active site of the cathepsins, the rest of the proteins were blocked as not being involved in the proteolytic function of cathepsins. In the case of MOG, the connecting tail of the protein to the cell membrane was blocked from interacting with the active site of cathepsins. ZDOCK then provided the top five predicted results as output PDB files. The first (top) prediction was then imported in the SPDB viewer package and energy minimized with the implemented GROMOS96 force field. The visual molecular dynamics package was used for visualization and structural alignment (as mentioned above).

qPCR array technology was used to screen for transcriptional changes in genes of interest to EAE (51, 52). The relative expression of 84 genes relating to myelination, T cell activation and signaling, adaptive immunity, cytokines, chemokines, inflammation, apoptosis, cell adhesion, cell stress, immune receptors, and immune/CNS-related transcription factors were examined in the lumbar spinal cord of WT and Cybb2/2mice using the mouse multiple sclerosis RT2 Profiler PCR array (SABiosciences). Murine lumbar spinal cords were snap-frozen in liquid N2 and total RNA was extracted using the phenol-based RNeasy lipid tissue mini kit (Qiagen). cDNA was synthesized from 600 ng RNA using the RT2 First Strand kit (Qiagen). The qPCR array was optimized on an Eppendorf Mastercycler gradient 2S (Qiagen) as per the manufacturer’s instructions. In brief, custom plates and buffers were used to optimize the thermocycler, cDNA from a single mouse of interest was used in a single array, and the results were verified using the Web-based PCR array data analysis (http://www. sabiosciences.com/pcrarraydataanalysis.php) and confirmed with the Microsoft Excel–based PCR array data analysis template (PAMM-125Z). All gene expression was quantified relative to five housekeeping genes (including b-actin and b2-microglobulin) and passed all internal control tests (genomic DNA contamination, RNA quality, PCR performance). Three independent experiments for each group (WT, 12 d/peak; Cybb2/2, 12 d/peak; WT/Cybb2/2, mock injected) were tested to allow for statistical analysis. To measure macrophage/microglial polarization to an M2 phenotype, expression levels of the M2-associated marker ARG-1 were measured by conventional qPCR. cDNA was made from lumbar spinal cord RNA (described above) using iScript reverse transcriptase supermix for RT-qPCR (Bio-Rad). All primers were at 300 nM, had a single melting curve, had efficiencies between 90 and 100%, and were designed or verified using Primer 3 (National Center for Biotechnology Information). 18S (forward, 59-AGTCGGCATCGTTTATGGTC-39, reverse, 59-CGCGGTTCTATTTTGTTGGT-39) was used as an internal control and did not vary across treatments, with the following PCR conditions (in a Bio-Rad iQ5 thermocycler): 95˚C for 5 min and 40 cycles of 95˚C for 30 s and 58˚C for 30 s. ARG-1 (forward, 59-AGGGTTACGGCCGGTGGAGAG-39, reverse, 59-CCCCTCCTCGAGGCTGTCCTTT-39) was quantified under the following PCR conditions: 95˚C for 3 min, 50 cycles of 95˚C for 15 s, and 60˚C for 60 s (31). Expression is presented relative to 18S and relative to the mock control samples.

Fluorometric measurement of cathepsin activities in vitro Measurements of cysteine cathepsin activities from lysosomal extracts were performed as described (7). In brief, lysosomes were magnetically isolated from iron/dextran-loaded BMMfs and lysed in 20 mM sodium acetate buffer containing 0.1% Tween 80 at pH 5.0 (46). The lysosomal extracts were added to 0.2 M potassium acetate buffer (pH 5.0) containing 1 mM cysteine/cystine redox buffer 600:1 (221–236 mV) (47), varying concentrations of hydrogen peroxide, and cathepsin-specific fluorogenic substrates, including cathepsin S substrate (Ac-KQKLR-AMC, Ex of 354 nm, Em of 442 nm, 0.645 mg/well; Anaspec), cathepsin L substrate (Ac-HisArg-Tyr-Arg-ACC, Ex of 380 nm, Em of 460 nm, 5 mg/well; Calbiochem/ Merck), and aspartic cathepsin D/E substrate (Mca-GKPILFFRLK(Dnp)r-NH2, Ex/Em of 328/393 nm, 1 mg/well; Anaspec)]. Fluorescent dequenching of the substrates was measured using a FLUOstar Optima fluorescent plate reader (BMG Labtech), or an EnVision multilabel reader (PerkinElmer) at 37˚C. Slopes of initial reaction rates were determined by curve-fitting applications in Microsoft Excel and expressed relative to untreated controls.

Cathepsin-mediated MOG35–55 cleavage Recombinant cathepsin L (0.25 mg, Novoprotein, Shanghai, China) and S (5 ng, EMD Millipore, Billerica, MA) were added to 0.2 M potassium acetate buffer (pH 5.0) containing 1 mM cysteine/cystine redox buffer 600:1 (221–236 mV) (47), varying concentrations of hydrogen peroxide, and 10 mg MOG35–55. Digestions were incubated for 15 min (cathepsin S) or 60 min (cathepsin L) at 37˚C in a Bio-Rad MyCycler thermal cycler (Bio-Rad, Mississauga, ON, CAN), followed by heat denaturation for 2 min at 90˚C. Peptides were separated by reverse-phase HPLC on a Supelco Ascentis Express C18 column (10 cm length, 2.7 mm pore) (SigmaAldrich) using a 2–50% acetonitrile gradient with 0.1% TFA. Peaks were collected and corresponding peptides identified by MALDI-TOF and/ or liquid chromatography–tandem mass spectrometry (Southern Alberta Mass Spectrometry Centre, Calgary, AB, Canada). The relative abundance of the peptides was quantified using the area under corresponding peaks. Abundance of the cleavage product MOG42–55 relative to MOG35–55 (corresponding destruction of the I-Ab binding region) was calculated using the following formula: RPA = (Px/Sx)/(P0/S0), where RPA indicates relative peptide abundance, Px indicates product (MOG42–55) of sample, Sx indicates substrate (MOG35–55) of sample, P0 indicates product (MOG42–55) without hydrogen peroxide, and S0 indicates substrate (MOG35–55) without hydrogen peroxide.

Computational analysis of I-Ab binding and protease cleavage of MOG The immune epitope database and analysis resource MetaSVMp (a quantitative binding affinity program that employs the Immune Epitope Database was used to compute relative affinities between I-Ab and regions of MOG1–125 as previously described (48). The inverse of the IC50 (nM 3

Statistics Unless indicated, statistical analyses were completed by one-way ANOVA (or unpaired Student t test) with a Tukey test or nonparametric equivalent (p , 0.05). When a Bartlett’s test for equal variances failed, data were transformed (natural log transformation) and reanalyzed. Analyses of all clinical data were done using a Kruskal–Wallis test (Dunn’s multiple comparisons). Statistical analyses of enzymatic activity assays (of lysosomal extracts and MOG42–55 abundance) were done by nonlinear regression (variable slope model). All statistical analyses were completed using GraphPad Prism software (GraphPad Software, La Jolla, CA).

Results A deficiency of NOX2 subunits influences patterns of Ag processing BMMfs from mice deficient in either gp91phox (Cybb2/2) or p47 phox (Ncf) subunits of NOX2 did not produce a measurable respiratory burst within the phagosome following the uptake of IgG-opsonized particles (Fig. 1A, 1B). Consistent with previous reports, we found that the absence of NOX2 activity promoted the reductive potential of the phagosome (as evidenced by the enhanced disulfide bond reduction of phagocytosed cystine), which in turn favored activities of the cysteine cathepsins (cathepsin B, S, and L) and led to a significant increase in overall phagosomal proteolysis (Fig. 1C–F, Supplemental Fig. 1A, 1B). As anticipated, NOX2 deficiency did not alter the rate or extent of phagosomal acidification, or rates of endo/phagocytosis (Supplemental Fig. 1C–F).


vascular cuffs; 3, extensive perivascular cuffing with extension into adjacent subarachnoid space and CNS parenchyma; 4, extensive perivascular cuffing with increasing subarachnoid and parenchymal inflammation. Demyelination scores were: 0, no demyelination; 1, a few scattered naked axons; 2, small groups of naked axons; 3, large groups of naked axons; 4, confluent foci of demyelination; 5, widespread demyelination.


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Next, we determined whether the NOX2-mediated inhibition of selected lysosomal proteases (the cysteine cathepsins) influenced the pattern of proteolysis and thus the relative abundance of peptide fragment products of phagocytosed Ag. To achieve this we used the model Ag HEL, heavily derivatized with Alexa Fluor 488 succinimidyl ester and covalently coupled to opsonized experimental particles. BMMfs derived from WT and NOX2-deficient mice were allowed to phagocytose and process the labeled protein cargo for 90 min. BMMfs were subsequently lysed and the composition of the fluorescent peptide products ,10 kDa was resolved by reverse phase HPLC. Although this experimental approach precludes the identification of the antigenic peptides generated (due to the overwhelming background of nonfluorescent endogenous peptides), it provides a snapshot of the peptides derived from the phagocytosed Ag that are potentially available for MHC-II binding within the endolysosomal system. The traces representing peptide composition generated from phagocytosed protein by BMMfs were highly conserved and reproducible within each genotype

(Supplemental Fig. 1G). Most peaks (representing peptide products) were common to both traces from WT and Cybb2/2 macrophages (Fig. 1G, Supplemental Fig. 1G). However, the relative abundance of many peptides (as determined by area under the curve for a given peak) differed markedly in the presence or absence of NOX2 (Fig. 1G). Additionally, a peak reliably produced by WT BMMØs was consistently undetectable in Cybb2/2 samples (Fig. 1G). These data support the hypothesis that NOX2 modulates not only the overall efficiency of phagosomal proteolysis, but also influences the relative abundance of different peptide epitopes potentially available for MHC-II binding and presentation. NOX2 activity influences MHC-II presentation in an Ag-specific, APC-specific manner Because NOX2 activity modified the pattern of phagosomal antigenic proteolysis and composition of available peptides, we investigated whether this could, in turn, affect the presentation efficiency of particular MHC-II–restricted epitopes. To achieve


FIGURE 1. Phagosomes in NOX2-deficient BMMfs have diminished production of ROS, elevated bulk proteolysis and cysteine cathepsin activity, and altered relative abundance of peptide products compared with WT. (A and B) Phagosomal respiratory burst, (C and D) total proteolytic activity (rate of substrate-liberated fluorescence from the particlebound fluorogenic substrate DQ green Bodipy albumin), (E and F) cysteine cathepsin activities, and (G) relative abundance of HEL cleavage products of WT and NOX2-deficient BMMfs (Cybb2/2 and Ncf) after phagocytosis of fluorometric experimental particles. (A, C, and E) Representative real-time traces where RFU are directly proportional to the degree of substrate oxidation or hydrolysis and are taken from 0 to 90 min after phagocytosis. (B, D, and F) The average rates (taken from 30 to 80 min after phagocytosis) of oxidation, total proteolytic activity, and cysteine cathepsin activity relative to the WT BMMf samples. (G) Relative abundance of peaks produced during HPLC analysis of peptides collected from lysed BMMfs (WT and Cybb2/2) that were incubated with IgG particle-restricted Alexa Fluor 488– HEL for 90 min. Relative peptide abundance is the area under the curve for each peak proportional to WT samples (see representative traces in Supplemental Fig. 1G). Data represent three independent experiments relative to WT control. (B, D, F, and G) Data are presented as means 6 SEM. *p , 0. 05 versus WT control (by ANOVA).

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NOX2-deficient I-Ab mice show protection from MOG-induced EAE Because NOX2-deficient BMMfs have a reduced ability to process MOG to generate the vastly I-Ab–immunodominant MOG35–55

FIGURE 2. NOX2 deficiency in BMMfs does not affect efficiency of presentation of preprocessed peptide Ag, but affects processing of protein Ag in an Ag-specific manner. WT, Cybb2/2, and/or Ncf BMMf were incubated for 6 h with (A) no peptide (NP), OVA323–339 (332–339; 10, 25 mg/ml), OVA (10, 25 mg/ml); (B) MOG35–55 peptide (35–55; 25 mg/ml), rMOG1–125 (rMOG; 10, 25 mg/ml), 3-mm IgG-opsonized silica particles (multiplicity of infection of three to five) covalently coupled with rMOG1–125 (rMOG Beads); (C) mouse myelin (25, 50 mg/ml); or (D) 1 mg/ ml HEL. Activation of the OVA323–339- and MOG35–55-specific OTII and 2D2 CD4+ T cells was determined by expression of CD69 after 16 h with the BMMf. Activation of CD4+ T cell HEL-specific hybridomas (Hb1.9 responding to HEL20–35, H30.44 responding to HEL31–47, H46.13 responding to HEL48–62, and B04 responding to HEL74–90; stably transfected with pNFATeGFP) was determined by eGFP expression. (A–C) Data represent five to six independent experiments, presented relative to the WT 25 mg/ml MOG35–55/OVA323–339 peptide control [or relative to WT myelin (C)]. (D) Data represent four to seven independent experiments and are presented relative to a maximum eGFP expression induced by 50 ng/ml PMA with 1 mg/ml ionomysin. Data are presented as means 6 SEM *p , 0.05 versus internal WT controls (by ANOVA).

peptide, we reasoned that NOX2-deficient I-Ab mice would be less susceptible to MOG-induced EAE. Indeed, following the induction of EAE using the peptide MOG35–55 and rMOG, both


this, we examined the efficiency of epitope-specific CD4+ T cell activation, using OTII and 2D2 transgenic T cell models that respond to the I-Ab immunodominant peptides derived from OVA and MOG, respectively. In brief, BMMfs and BMDCs derived from WT and NOX2-deficient mice were allowed to endocytose unprocessed whole Ag or preprocessed antigenic peptides for a defined period. The activation of CD4+ T cells isolated from OTII and 2D2 mice and coincubated with the APCs were assessed by measuring surface expression of the early activation markers CD69 and CD25 as previously described (15, 25, 31, 33, 35, 53, 54). Deficiency in either the gp91 or p47 subunits of NOX2 did not affect the presentation of either model Ags when APCs were pulsed with preprocessed peptide (Fig. 2A, 2B, Supplemental Fig. 2A, 2C, 2F–H). Similarly, WT and NOX2-deficient APCs were equally able to activate OVA323–339-specific CD4+T cells when given whole OVA (Fig. 2A, Supplemental Fig. 2F). In contrast, however, the ability of BMMfs, but not BMDCs, to activate MOG35–55-specific CD4+T cells in the absence of NOX2 was significantly diminished when given the whole recombinant MOG protein (rMOG, MOG1–125) (Fig. 2B, Supplemental Fig. 2G, 2H). The discrepancy between WT and NOX2-deficient BMMfs to present MOG35–55 was further increased when NOX2 activity was enhanced by delivering the rMOG protein on IgG-opsonized experimental particles (Fig. 2B, Supplemental Fig. 2H). To determine whether the processing of endogenously derived MOG would be similarly affected by the NOX2 activity of macrophages, myelin sonicates from whole mouse brain were prepared and given to BMMfs. Similar to findings with recombinant MOG, NOX2-deficient macrophages were significantly less able to process and present MOG35–55 from endogenously expressed MOG in purified myelin (Fig. 2C, Supplemental Fig. 2E, 2I). Collectively, these data suggest that NOX2 activity selectively affects the presentation of Ag through specific modulation of Ag processing. To further interrogate the ability of NOX2 to alter processing patterns of a single Ag, we measured the efficiency of WT and Cybb2/2 BMMfs to process four distinct HEL epitopes (HEL20–35, HEL31–47, HEL48–62, and HEL74–90) from whole HEL protein and present them to epitope-specific CD4+ T cell hybridomas (Hb1.9, H30.44, H46.13, and B04, respectively) (26). To achieve this, T cell hybridomas were first stably transfected with an NFAT-enhanced IL-2 promoter–driven eGFP construct, followed by multiple rounds of selection by FACS to enrich those hybridoma clones that expressed eGFP only following presentation of their cognate peptide epitope by APCs (25). When added to BMMfs that were previously pulsed with HEL protein, we found that WT and Cybb2/2 BMMfs were equally able to induce eGFP expression in two of the hybridomas (Hb1.9 and H46.13), indicating that the processing/presentation efficiency of HEL20–35 and HEL48–62 peptides were unaffected by NOX2 (Fig. 2D). Interestingly, NOX2 had a disparate effect on the processing/presentation efficiencies of the two other HEL epitopes, with Cybb2/2 BMMfs showing compromised presentation of HEL31–47 (reduced eGFP expression in H30.44), but an enhanced presentation efficiency of HEL74–90 (increased eGFP expression in B04) (Fig. 2D). Consistent with earlier biochemical findings (Fig. 1), these data indicate that NOX2 does not uniformly affect processing of a given Ag, but differentially affects relative epitopic generation from a single Ag.


The Journal of Immunology Cybb2/2 and Ncf mice showed a reduced incidence and a delayed onset of clinical disease, as well as a reduction in the average of peak clinical scores (Fig. 3A–E) when compared with WT mice. Supporting the clinical observations, NOX2deficient mice were shown to have on average 2- to 3-fold fewer infiltrating macrophages, microglia, B cells, CD4+ T cells, and CD8+ T cells in spinal cord tissue at 12 d postinoculation as compared with WT (Fig. 3F, 3G). NOX2 deficiency also resulted in significantly lower numbers of MOG 35–55 tetramer+ CD4+ T cells within spinal cord tissue, as well as reduced CD4+ T cell expression of IFN-g, indicating reduced MOG-specific T cell activation within the CNS in the absence of NOX2

4995 (Fig. 3H). Interestingly, although NOX2 deficiency also afforded some protection from full-length MOG protein-induced EAE, it was typically less apparent, and the onset of EAE in Ncf mice was not significantly delayed (Fig. 3E). We reasoned that this may result from the generation of other potential immunogenic epitopes (MOG71–90 and MOG101–120) in these mice, even though we found them to be insufficient to induce EAE on their own. Nonetheless, consistent with the findings that NOX2-deficient BMMfs are less efficient in the generation of the I-Ab–immunodominant MOG epitope MOG35–55 in vitro, NOX2-deficient I-Ab mice show protection from MOG-induced, particularly MOG35–55induced, EAE.


FIGURE 3. NOX2-deficient mice are partially protected from EAE. Clinical disease course of NOX2-deficient (Cybb2/2 and Ncf) and WT mice after active induction of EAE with (A) 50 mg MOG35–55 or (B) 200 mg rMOG1–125. (C) Clinical incidence (all mice that exceed a clinical score of 0), (D) maximum clinical score, and (E) time to clinical onset (score of at least 0.5) of EAE induced using 50 mg MOG35–55 (35–55) or 200 mg rMOG1–125 (rMOG). (A–E) Mice were clinically scored (0–4) daily for 40 d. Data represent four independent experiments, each containing cohorts of three to seven mice per genotype (total mouse numbers: MOG35–55, n = 25 WT, 23 Cybb2/2, 14 Ncf, rMOG1–125 n = 28 WT, 19 Cybb2/2, 19 Ncf); (F–H) n = 3–4 per genotype/injection. (F) The total number of leukocytes isolated from the spinal cord (via a discontinuous Percoll gradient) of mice 12 d postinoculation with 50 mg MOG35–55 (35–55), 200 mg rMOG1–125 (rMOG), or CFA alone (Mock). (G) The number of macrophages (Mf, CD11b+/CD45high), microglia (MG, CD11b+/CD45 low), B cells (B220+/CD45+), CD8+ T cells (CD8+/CD3+), and CD4+ T cells (CD4+/CD3+) isolated from the spinal cord of mice 12 d postinjection with 50 mg MOG35–55. (H) The number of infiltrating CD4+ T cells that express Foxp3, IFN-g, IL-17, (CD4+/Foxp3, CD4+/IFN-g, CD4+/IL17, respectively) or MOG35–55/I-Ab–positive CD4+ T cells (CD4+/MOG35–55 tetramer+) isolated from spinal cord of mice 12 d after inoculation with 50 mg MOG35–55. Data are presented as means 6 SEM. *p , 0.05 versus WT internal control (clinical data, Kruskal–Wallis; Student t test).

4996 Clinical progression after onset and the final pathology of EAE in clinically affected animals were not influenced by NOX2 deficiency

FIGURE 4. NOX2 deficiency does not alter disease severity of EAE in clinically affected mice. (A) The maximum clinical score of mice that reached a clinical score of 0.5 for at least 1 d after injection with 50 mg MOG35–55 (35–55) or 200 mg rMOG1–125 (rMOG). Data represent four independent experiments, each containing cohorts of three to seven mice per genotype. (B) The number of macrophages (Mf, CD11b+/CD45high), microglia (MG, CD11b+/ CD45low), B cells (B220+/CD45+), CD8+ T cells (CD8+/CD3+), and CD4+ T cells (CD4+/CD3+) isolated from spinal cord of mice at the peak of the disease following inoculation with rMOG1–125 counted and characterized by flow cytometry (n = 3–4). (C and D) Histopathology of CNS removed from mice immunized with MOG35–55 at the peak of the disease (score .2.5). (C) Representative micrographs of lumbar spinal cord stained with Luxol fast blue (LFB) or H&E; arrows indicate areas of inflammation and demyelination. (D) Histological scores for inflammation and demyelination (n = 3). (E) Peripheral activation and peptide-driven ex vivo expansion of lymphocytes. Lymph node cells were isolated from inguinal lymph nodes of mice 12 d postinoculation with rMOG/CFA and restimulated in vitro with 25 mg/ml MOG peptides 35–55, 71–90, and 101–120 for 48 h. Stimulation index represents [3H]thymidine incorporation as measured by scintillation (n = 6). Data are presented as means 6 SEM; significant differences. *p , 0.05 versus WT internal control (clinical data, Kruskal– Wallis; by ANOVA).

MOG35–55 CD4+ T cell clones in the peripheral lymph nodes of WT and Cybb2/2 mice were measured 12 d postinoculation. Using a MOG35–55-specific tetramer and peptide-driven ex vivo proliferation, it was found that the clonal expansion of MOG35–55responsive CD4+ T cells in secondary lymphoid organs was unchanged in Cybb2/2 mice following inoculation with MOG35–55 peptide (Supplemental Fig. 3D). Because DCs are primarily charged with the activation and expansion of naive CD4+ T cells, this finding is consistent with the ability of NOX2-deficient BMDCs to efficiently present MOG35–55 (Supplemental Fig. 2G). Interestingly, although the peripheral expansion of CD4+ T cells specific to the immunodominant MOG35–55 was equivalent, Cybb2/2, but not WT, mice exhibited additionally expanded MOG71–90- and MOG101–120-responsive T cell clones (Fig. 4E, Supplemental Fig. 3E). This finding suggests that while NOX2 deficiency does not alter the generation of MOG35–55 in DCs, it actually enhances both the generation and presentation of the alternative MOG epitopes, further supporting a role of redoxmediated modification to patterns of Ag processing in an in vivo setting. Nonetheless, because the initial priming of MOG35–55 CD4+


Although Cybb2/2 and Ncf mice show reduced incidence and delayed onset of EAE, we noted that NOX2-deficient animals that did develop EAE symptoms progressed (albeit delayed) in a manner that was clinically indistinguishable from affected WT mice (Fig. 4A). Likewise, mice that were sacrificed at the peak of the clinical disease (between clinical scores of 2.5 and 3.5) exhibited comparable levels of leukocyte infiltration, inflammation, and demyelination of the spinal cord (Fig. 4B–D), peripheral cytokines (Supplemental Fig. 3A), proliferative response to MOG35–55 (Fig. 4E), ARG-1 mRNA levels (Supplemental Fig. 3B), and general CNS mRNA expression patterns of genes relevant to EAE (Supplemental Fig. 3C). These findings suggest that NOX2 did not act to modify the progression of disease once it had passed the clinically apparent threshold, but it influenced the stages of pathogenesis that preceded the establishment of overt encephalomyelitis. To determine whether peripheral responses to the MOG inoculation were affected by NOX2, the degrees of expansion of



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T cells in response to the inoculation of exogenous MOG was unchanged by NOX2, we reasoned that the inefficiency of NOX2-deficient macrophages to process endogenous MOG in the CNS limited the progression to clinically apparent encephalomyelitis. Reactivation of MOG35–55-specific CD4+ T cells within the CNS during preclinical EAE is compromised in NOX2-deficient mice

Lysosomal cysteine cathepsins destroy critical regions in MOG35–55 in a redox-dependent manner We hypothesized that the inefficiency of NOX2-deficient macrophages to present the immunodominant MOG35–55 peptide would be due to the increased hydrolysis of critical regions within MOG35–55 by cysteine cathepsins in the early phagosome. Using SitePredict, a sequence-based prediction tool for protease cleavage, we identified two potential cathepsin L/S cleavage sites within the critical I-Ab binding region of MOG35–55 (Arg41/Ser42 and Ser45/Arg46) (49, 50, 55, 56). Pertinently, both of these cleavage sites are located on a solvent-accessible loop in the MOG protein, rendering them potentially susceptible to hydrolysis in the early phagosome. This prediction was further supported by determining the spatial interactions between the MOG protein and cathepsins L and S using a ZDOCK algorithm (44, 45). ZDOCK makes predictions on interactions between two protein structures based on shape complementarity, desolvation free energy, and electrostatic energies. By constraining the protein–protein interaction based on the location of the cathepsin active sites, ZDOCK has also predicted that the loop region of amino acid 41–46 on the MOG can intimately interact with the active sites of both cathepsins L and S (Fig. 6A, 6B). Using recombinant systems and mass spectral analysis, we confirmed that both cathepsins L and S efficiently degrade MOG35–55 under reductive conditions within the critical 41–46 region (Fig. 6C, 6D). Because both cathepsin L and cathepsin S are oxidatively inhibited by NOX2 products (Fig. 6E), we further demonstrated that MOG35–55 was protected from cleavage by cathepsins L and S by increasing concentrations of hydrogen peroxide (Fig. 6D). Taken together, these predictions and experimental data demonstrate that the I-Ab binding region of MOG35–55 in the native protein is able to be specifically and efficiently cleaved by cathepsin L and cathepsin S under reductive, but not oxidative, conditions.

Discussion In this study, we have shown that NOX2, in addition to its antimicrobial function, alters the pattern of phagolysosomal processing

FIGURE 5. The efficiency of reactivation of MOG35–55-specific 2D2 CD4+ T cells is diminished within the CNS NOX2-deficient mice. (A and B) Transgenic MOG35–55-specific 2D2 CD4+ T cells were isolated and expanded ex vivo using IL-12 and MOG35–55 for 72 h before adoptive transfer into WT and Cybb2/2 mice 7 d after inoculation with MOG35–55. Transferred MOG35–55-specific 2D2 CD4+ T cells were isolated from the CNS after 72 h in vivo using a discontinuous Percoll gradient and identified (CD4+, Vb11+, Va3.2+) by flow cytometry. Expression of CD25 (CD4+, Va3.2+, CD25+) and IFN-g (CD4+, Va3.2+, IFNg+) by transferred MOG35–55-specific CD4+ T cells were determined by flow cytometry. (C and D) Corresponding flow cytometry plots. For (A) and (B), n = 5. Data are presented as means 6 SEM. *p , 0.05) (by Student t test).

of MHC-II Ags in an Ag- and cell-specific manner. Furthermore data presented strongly suggest that this phenomenon is mediated through a redox-based mechanism, where phagosomal NOX2 inhibits local cysteine cathepsins which require a reductive environment for optimal activity. Specifically, we found that the I-Ab– immunodominant epitope of MOG (MOG35–55), which contains a cathepsin L/S cleavage site within the I-Ab binding region, is not efficiently presented by NOX2-deficient macrophages. Additionally, we demonstrated that NOX2-deficient mice were partially


To interrogate whether CNS-resident NOX2-deficient APCs are compromised in their ability to present endogenous MOG in preclinical EAE, the efficiency of reactivation of adoptively transferred MOG35–55-specific 2D2 CD4+ T cells in WT and Cybb2/2 mice was examined. 2D2 CD4+ T cells were expanded in vitro and transferred into WT and Cybb2/2 mice 7 d after inoculation with MOG35–55. CNS tissue was recovered 72 h after transfer, and recruitment and reactivation MOG35–55-specific 2D2 CD4+ T cells were determined by flow cytometry. As expected during this preclinical phase, MOG35–55-specific 2D2 CD4+ T cells (Va3.2+ and Vb11+ CD4+ T cells) were recruited in equivalent numbers to the CNS in WT and Cybb2/2 mice (Fig. 5A). However, these recruited MOG-responsive CD4+ T cells displayed significantly reduced expression of the activation marker CD25 and the Th1 effector IFN-g in the CNS of Cybb2/2 mice demonstrating a reduced MOG35–55-specific reactivation in the absence of NOX2 in this tissue (Fig. 5B–D).

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protected from MOG-induced EAE, a CD4+ T cell–driven autoimmune disease, despite eliciting a normal peripheral immune response to the inoculated MOG Ag. Hence, we propose that the partial protection from EAE in NOX2-deficient mice is brought about by ineffective reactivation of effector CD4+ T cells within the CNS due to the inefficient processing of endogenous MOG

to the immunodominant MOG35–55 epitope by NOX2-deficient macrophages (Fig. 7). The effect of NOX2 on MOG processing appeared to be APCspecific. We found that when APCs were given intact MOG, NOX2 activity enhanced MOG35–55 presentation in BMMfs, but had no effect in BMDCs (Supplemental Fig. 2G). It could be argued that


FIGURE 6. MOG is cleaved within the MOG35–55 I-Ab binding region by cathepsin L and S in a H2O2-inhibitable manner. (A and B) Novel representations of the spatial interaction between cathepsin L/S and MOG as determined by a ZDOCK algorithm. The complex is colored based on protein secondary structure: yellow denotes b-sheets; purple denotes a helix; blue denotes 310 helix. The region of residues 35–55 on MOG has been highlighted in green. Target residues within the predicted cleavage site of cathepsin L/S on MOG are shown with sticks. Cyan, blue, red, yellow, and white sticks represent carbon, nitrogen, oxygen, sulfur, and hydrogen atoms, respectively. (A) The inset presents a magnified view of the predicted target loop (Arg41, Ser42, Pro43, Phe44, Ser45, and Arg46) of MOG within the active site of cathepsin L (Cys25 and His163). (B) The inset presents a magnified view of the predicted target loop (Arg41, Ser42, Pro43, Phe44, Ser45, and Arg46) of MOG within the active site of cathepsin S (Cys25 and His164). (C) A schematic of the predicted and experimentally confirmed cathepsin L/S cleavage site in relation to the I-Ab binding region (red) of the immunodominant epitope of MOG (MOG35–55), and the corresponding cleavage products that were identified by MALDI-TOF MS. (D) The ability of recombinant cathepsin L and recombinant cathepsin S to cleave MOG35–55 was assessed using MOG35–55 in a reconstituted system with increasing concentrations of H2O2. Following incubation at 37˚C, relative quantities of remaining substrate (MOG35–55) and cleavage products (MOG42–55 and MOG35–41) were quantified by reverse-phase HPLC following peak identification by MALDI-TOF MS. Data are presented as the abundance of the cleavage product MOG42–55 relative to MOG35–55 (corresponding destruction of the I-Ab binding region) and was calculated using the following formula: RPA = (Px/Sx)/(P0/S0), where RPA indicates relative peptide abundance; Px indicates product (MOG42–55) of sample, Sx indicates substrate (MOG35–55) of sample, P0 indicates product (MOG42–55) without hydrogen peroxide, and S0 indicates substrate (MOG35–55) without hydrogen peroxide. Cathepsin L (IC50 of 35.97 mM H2O2; R2 = 0.91) and cathepsin S (IC50 of 60.84 mM H2O2; R2 = 0.99). (E) Relative rates of hydrolysis of cathepsin-specific fluorogenic substrates (cathepsin L, Ac-HRYR-ACC; cathepsin S, Ac-KQKLR-AMC; cathepsin D/E, Mca-GKPILFFRLK-Dnp) by BMMf lysosomal extracts in increasing concentrations of H2O2. The cathepsin L (IC50 by BMMf lysosomal extracts of 42.34 mM H2O2; R2 = 0.96), cathepsin S (IC50 of 94.28 mM H2O2; R2 = 0.97), and aspartic cathepsin D/E cleavage (cathepsin D/E: does not converge). Data are from three experiments and presented as means 6 SEM. Statistical analysis was performed using nonlinear regression (variable slope model) where the inhibitor (H2O2) is log-transformed.


FIGURE 7. An illustration of the reactivation of effector Th cells in the CNS of WT and NOX22/2 mice from endogenous MOG. MOG-containing myelin is phagocytosed during CNS inflammation and processed within phagosomes. In WT macrophages, NOX2 limits the activities of cathepsin L and S and these macrophages can efficiently present MOG35–55 to MOG35–55-specific effector Th cells. In a feed-forward response, the reactivated Th cells further recruit and activate macrophages, leading to increased phagocytosis of myelin and upregulation of NOX2 and MHC-II. NOX2-deficient macrophages cannot oxidatively inactivate cathepsin L and S within the phagosome, and thus have a higher likelihood of cleaving the immunodominant MOG35–55 in the I-Ab binding region. This prevents efficient MHC-II presentation to MOG35–55-specific effector Th cells and the efficient perpetuation of the autoimmune response. The MOG35–55/I-Ab complex is adapted from Carrillo-Vico et al. (50).

faithfully recapitulate the antigenic repertoires produced by DCs in the primary immune response. Although we have shown that NOX2-deficient macrophages are unable to efficiently process and present MOG and to efficiently reactivate MOG35–55-responsive CD4+ T cells in the CNS, it is impossible to definitively exclude other effects that NOX2 deficiency may have during EAE. Extracellular release of NOX2derived ROS by infiltrating leukocytes in NOX2-competent mice could potentially contribute to the pathology of EAE, which would manifest as a comparatively milder clinical syndrome in NOX2-deficient mice. In our study, whereas the incidence and onset of clinically apparent EAE was altered in NOX2-deficient mice, animals that did succumb to EAE displayed identical clinical courses (following initial onset clinical signs) and the same severity of disease (as determined by maximum clinical score, histopathological findings, cellular infiltration, and CNS transcriptional analysis) irrespective of genotype. Although the role of extracellular ROS-driven pathology cannot be excluded, our observations do not fit with this model. Another possible contributing factor could arise through an absence of NOX2-mediated signaling, which may polarize macrophages toward a protective M2 (alternatively activated) phenotype. Specifically, there are two reports of a deficiency in p47phox (Ncf) potentiating an M2-like phenotype in peritoneal macrophages or microglia in response to Listeria monocytogenes or LPS, respectively (61, 62). Interestingly, macrophages deficient in gp91phox (Cybb2/2) did not show the same propensity for M2 polarization (61). During peak EAE, we found that the M2 marker Arg-1 within the CNS was equally expressed in NOX2-deficient and WT mice (Supplemental Fig. 3B), and that Th1 response predominated in both draining lymph nodes and CNS tissues in response to MOG inocula, irrespective of genotype (Supplemental Fig. 3C, 3D). Additionally, mice deficient in either p47phox (Ncf) and gp91phox (Cybb2/2) displayed similar levels of protection from EAE (Fig. 3), as well as a similar pathology to WT mice at the peak of disease (Fig. 4). Taken together, these data suggest that the primary protection from EAE by NOX2 deficiency is unlikely to be the result of


BMDC preparations may not be homogeneous and therefore we cannot draw absolute conclusions regarding the effect of NOX2 on MOG processing in DCs from these experiments. However, the differential effect of NOX2 on APC processing was supported by the finding that following inoculation with full-length MOG, NOX2-deficient mice induced a normal MOG35–55-reactive primary T cell response in the lymph node, but a reduced secondary response and reactivation of expanded MOG35–55-reactive CD4+ T cells in the CNS (Figs. 3, 5, Supplemental Fig. 3A, 3D). The disparity between these related cell types may be explained by lower NOX2 activity in DCs or by their enhanced processing and presentation efficiency of low abundance Ags (1, 45). Alternatively, this disparity may be the result of the differential expression of cysteine cathepsins between these APCs. Although cathepsin L mRNA and protein is detectable in DCs, cathepsin L activity is notably absent (43, 57, 58). Macrophages, alternatively, express high levels of the active form of cathepsin L (4, 41). Similarly, expression of active cathepsin S is modest in DCs but highly expressed in macrophages, particularly in response to classical activating stimuli (1, 59). Because the I-Ab-binding region of MOG35–55 is susceptible to cleavage by cathepsins L and S (Fig. 6), it stands to reason that the regulation of these enzymes by NOX2 is more important to epitopic preservation in macrophages than in DCs. Nevertheless, differential representation of presented peptide epitopes on DCs and macrophages presents an interesting quandary. Whereas DCs are typically charged with priming the CD4+ T cell response to a new Ag, macrophage presentation to expanded CD4+ T cells at sites of inflammation is often essential for the propagation and escalation of the effector immune responses to persistent Ag (42, 60). Our data indicate that, in NOX2-deficient mice, lymph node DCs present an MHC-II–restricted peptide epitope that cannot be efficiently recreated by peripheral macrophages from endogenous antigenic sources (e.g., myelin), and this ultimately creates an inefficiency between priming and effector Th responses. Hence, in this scenario, NOX2 activity within activated tissue macrophages acts not only to slow the overall rates of phagosomal proteolysis, but also to more

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Acknowledgments We thank Jason Lemmon, Sibapriya Chaudhuri, and Dr. Joanna Rybicka for their logistical support. We also thank Bill Zaun for artistic services.

Disclosures The authors have no financial conflicts of interest.

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decreased ROS-mediated pathology or an undefined role of p47 in myeloid polarization, and that their contribution to the observed phenotype is subtle, if at all. This is additionally supported by Hultqvist et al. (63) who reported that p47-deficient H2q mice developed EAE with the same incidence as WT H2q mice following induction with MOG1–125, but not with MOG79–96. This further indicates that protection from MOG-induced EAE in NOX2-deficient mice is an Ag-, haplotype-specific phenomenon. The possible binding regions of the MOG protein to I-Ab (with reasonable affinity) are strikingly limited to MOG35–55 (actual I-Ab binding at ∼MOG40–46), making this region ultimately immunodominant in this haplotype (Supplemental Fig. 3F) (50, 64). The finding that the residues 41–42 are particularly susceptible to cathepsin L (in a reductive microenvironment) (Fig. 6C, 6D) may not only underpin the disconnect between the abilities of DCs and macrophages to activate T cells in NOX2-deficient mice, but may also give insight into why MOG escapes tolerization in I-Ab mice. Because thymic epithelial cells express high levels of cathepsin L (43), efficient cleavage within MOG40–46 may lead to inefficiencies of MOG35–55 presentation and limit the deletion of MOG-autoreactive CD4+ T cells. Indeed, the susceptibility of myelin basic protein to asparagine endopeptidase led Manoury et al. (65) to propose a similar mechanism of autoreactivity of myelin basic protein in certain haplotypes of mice. The relationship between levels of proteolysis and efficiency of Ag presentation has been the topic of many studies during the past decade (1, 2, 7, 66). However, most attention has been paid to determining the efficiency of processing rather than the patterns of processing. Because the Th arm of the immune system relies on the faithful reproduction of MHC-II–restricted peptides from any given Ag during education, activation, and effector function (all performed by different APCs), subtle changes to Ag processing chemistries could lead to a breakdown in tolerance or conversely deficiencies in the Th cell response. Interestingly, NOX2 deficiencies in humans (chronic granulomatous disease) have been associated with altered susceptibilities to a number of autoimmune disorders, including systemic lupus erythematous, rheumatoid arthritis, colitis, and Crohn’s disease (67–73). In this study, we demonstrate that the redox state of phagosomes is a determinant of Ag processing fidelity and its modification by NOX2 can ultimately impact Th cell–driven disease processes.



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Major Histocompatibility Complex (MHC) Class I and MHC Class II Proteins: Conformational Plasticity in Antigen Presentation.

Early BCR Events and Antigen Capture, Processing, and Loading on MHC Class II on B Cells.

ATGs help MHC class II, but inhibit MHC class I antigen presentation.

Electroporation of exogenous antigen into the cytosol for antigen processing and class I major histocompatibility complex (MHC) presentation: weak base amines and hypothermia (18 degrees C) inhibit the class I MHC processing pathway.

Chemical approaches to discovery and study of sources and targets of hydrogen peroxide redox signaling through NADPH oxidase proteins.

MHC class II-restricted presentation of intracellular antigen.

Recent advances in Major Histocompatibility Complex (MHC) class I antigen presentation: Plastic MHC molecules and TAPBPR-mediated quality control.

MHC class II antigen presentation by dendritic cells regulated through endosomal sorting.

Viral immune evasion: Lessons in MHC class I antigen presentation.

Long-Term Effects of Maternal Deprivation on Redox Regulation in Rat Brain: Involvement of NADPH Oxidase.

Giving CD4+ T cells the slip: viral interference with MHC class II-restricted antigen processing and presentation.

Pseudomonas aeruginosa Cif protein enhances the ubiquitination and proteasomal degradation of the transporter associated with antigen processing (TAP) and reduces major histocompatibility complex (MHC) class I antigen presentation.

Cancer-Associated Oxidase ERO1-α Regulates the Expression of MHC Class I Molecule via Oxidative Folding.

Processed antigen binds to newly synthesized MHC class II molecules in antigen-specific B lymphocytes.

Trichostatin A, a histone deacetylase inhibitor suppresses NADPH Oxidase 4-Derived Redox Signalling and Angiogenesis.

NADPH oxidase-dependent redox signaling in TGF-β-mediated fibrotic responses.

Signalling through the MHC class II cytoplasmic domain is required for antigen presentation and induces B7 expression.