Abstract

   While oxidative stress has been linked to multiple sclerosis (MS), the
   role of superoxide-producing phagocyte NADPH oxidase (Nox2) in central
   nervous system (CNS) pathogenesis remains unclear. This study
   investigates the impact of Nox2 gene ablation on pro- and
   anti-inflammatory cytokine and chemokine production in a mouse
   experimental autoimmune encephalomyelitis (EAE) model. Nox2 deficiency
   attenuates EAE-induced neural damage and reduces disease severity,
   pathogenic immune cells infiltration, demyelination, and oxidative
   stress in the CNS. The number of autoreactive T cells, myeloid cells,
   and activated microglia, as well as the production of cytokines and
   chemokines, including GM-CSF, IFNγ, TNFα, IL-6, IL-10, IL-17A, CCL2,
   CCL5, and CXCL10, were much lower in the Nox2^−/− CNS tissues but
   remained unaltered in the peripheral lymphoid organs. RNA-seq profiling
   of microglial transcriptome identified a panel of Nox2 dependent
   proinflammatory genes: Pf4, Tnfrsf9, Tnfsf12, Tnfsf13, Ccl7, Cxcl3, and
   Cxcl9. Furthermore, gene ontology and pathway enrichment analyses
   revealed that microglial Nox2 plays a regulatory role in multiple
   pathways known to be important for MS/EAE pathogenesis, including
   STAT3, glutathione, leukotriene biosynthesis, IL-8, HMGB1, NRF2,
   systemic lupus erythematosus in B cells, and T cell exhaustion
   signaling. Taken together, our results provide new insights into the
   critical functions performed by microglial Nox2 during the EAE
   pathogenesis, suggesting that Nox2 inhibition may represent an
   important therapeutic target for MS.

   Keywords: NADPH oxidase, reactive oxygen species, experimental
   autoimmune encephalomyelitis, chemotaxis, microglia

Introduction

   Multiple sclerosis (MS) is one of the major neurodegenerative diseases.
   Many drugs are available for MS therapy, but these treatments often do
   not effectively halt disease progression. Thus, the identification of
   disease-modifying drugs that can stop MS progression are urgently
   needed ([39]1). Mouse experimental autoimmune encephalomyelitis (EAE)
   is widely used to model human MS ([40]2). The key features of the mouse
   EAE model that somewhat recapitulate the several immunopathological and
   neuropathological features of human MS include inflammation,
   demyelination, axonal loss, and gliosis ([41]3). While the roles
   performed by immune cells related to autoimmune functions [e.g.
   autoreactive CD4^+ T cells (T[H]1 and T[H]17 cells), CD8^+ T cells,
   memory B cells, and myeloid cells (monocytes, dendritic cells)] are
   well known ([42]4), the influence of microglia on MS/EAE remains
   unclear.

   Microglia are a major source of pro-inflammatory cytokines (e.g. TNFα,
   IL-1β, IL-6, IL-17, and IL-23) and chemokines (e.g. CCL2, CCL3, CCL4,
   CCL5, CCL12, and CCL22) that may worsen MS/EAE ([43]5). Therefore,
   deregulated microglia can aggravate devastating demyelination and
   neuronal damage in the brains of MS patients ([44]6). Microglial
   nodules, characterized by an absence of leukocyte infiltration,
   astrogliosis, or demyelination, in normal-appearing white matter of MS
   patient brains are linked to the earliest stage of MS lesion formation.
   Known as “pre-active lesions,” these clusters of activated microglia
   are believed to eventually develop into pronounced and active
   demyelinating MS lesions ([45]7). Kinetics studies from EAE mouse
   models suggest that microglia are immediately activated by MOG
   treatment and represent the earliest cell population to take up myelin
   antigens ([46]8). Subsequently, through major histocompatibility
   complex (MHC) molecules, these microglia re-stimulate and recruit
   autoreactive T cells into the central nervous system (CNS) ([47]9).

   Microglia-generated superoxide and its immune cell-produced metabolites
   play a critical role in the progression of various neurodegenerative
   disorders, including MS/EAE ([48]10, [49]11).
   Nicotinamideadenine-dinucleotide phosphate (NADPH) oxidase (Nox2) is a
   key superoxide-producing enzyme that forms reactive oxygen species
   (ROS) ([50]12). Nox2 is highly expressed in professional phagocytes
   (e.g. neutrophils, monocytes, macrophages, microglia, and dendritic
   cells). Nox2 is critical for host defense because it produces reactive
   oxidants, activates granular proteases, and assists in the generation
   of neutrophil extracellular traps. Nox2 is the catalytic,
   membrane-bound subunit of NADPH oxidase. It is composed of an
   N-terminal transmembrane domain that interacts with two heme groups and
   a C-terminal domain that associates with both flavin adenine
   dinucleotide and NADPH ([51]13). Nox2 remains inactive until it
   interacts with the membrane-anchored p22phox, resulting in the
   formation of the flavocytochrome b558 heterodimer ([52]14). After
   activation by various infectious agents, the regulatory subunits
   p67phox, p47phox, p40phox, and the small Rho GTPase Rac are recruited
   to the complex to form NADPH oxidase on the plasma or phagosomal
   membrane ([53]15). Here, Nox2 converts molecular oxygen into superoxide
   anions, which can be further transformed into antimicrobial metabolites
   (e.g. hydroxyl anion and hydrogen peroxide) ([54]16). Thus, Nox2 is a
   major contributor to oxidative stress and subsequent neurotoxicity in
   the CNS ([55]17). However, the mechanisms underlying Nox2-dependent MS
   pathogenesis remain unknown. Here, we sought to address this gap in our
   understanding of MS/EAE pathogenesis by investigating the contribution
   of Nox2 activity to myelin oligodendrocyte glycoprotein (MOG)-elicited
   EAE.

   In this study, we provide strong evidence indicating that microglia
   play a critical role in the induction of EAE pathologies.
   Nox2-generated superoxide and excessive oxidative stress as well as
   Nox2-mediated chemotaxis in microglia are both essential mediators to
   cause advanced demyelination of oligodendrocytes and damage of neurons.
   Furthermore, our data also suggests Nox2 is prime target for developing
   therapy for MS/EAE.

Materials and Methods

Animals

   C57BL/6 gp91^phox−/− [Nox2 KO mice (gp91^Cybbtm1Din/J)] mice ([56]18)
   were kindly provided by Dr. Chi-Chang Shieh (Institute of Clinical
   Medicine, National Cheng Kung University College of Medicine, Tainan
   city, Taiwan). Control mice, C57BL/6 gp91^phox+/+ (wildtype mice), were
   purchased from National Laboratory Animal Center in Taipei City,
   Taiwan. Both mouse strains were subsequently bred at the Animal Center
   of the National Defense Medical Center under pathogen-free conditions.
   Mice were treated in accordance with the Institutional Animal Care and
   Use Committee of the National Defense Medical Center guide for
   experiments and approved by the committee in the same office (IACUC:
   19-058).

Chronic EAE Induction and Disease Score

   Mice were given a subcutaneous injection of 100 μg of MOG[35–55]
   emulsified with complete Freund’s adjuvant (MOG/CFA) which contained
   0.8 mg of heat-inactivated Mycobacterium tuberculosis (H37RA; Difco
   Laboratories) in their flanks. Each animal also received pertussis
   toxin ([57]19) (500 ng i.p.; List Biological Laboratories) on day 0 and
   day 2 post-immunization. Mice were monitored daily up to 42 days for
   signs of disease. The EAE clinical score was determined based on the
   following scale: 0: normal mouse, no overt signs of disease; 1: limp
   tail or hind limb weakness, but not both; 2: limp tail and hind limbs
   weakness or poor balance or head tilting when walking; 3: Limp tail and
   complete paralysis of hind limbs or one front and one hind limb; 4:
   Limp tail, complete hind limbs and partial front limbs paralysis; 5:
   moribund state or death. For our pathologic score, the scale ranges
   from 0 to 5 and represents normal to most severe immune cells’
   infiltration at the myelin area. 0: no infiltration, 1: ≤10%, 2:
   11–20%, 3: 21–30%, 4: 31–40%, 5: >40% (reviewed by a neuropathologist).
   In-between scores (i.e. 0.5, 1.5, 2.5, 3.5, 4.5) when the clinical or
   pathologic picture lies between two defined scores.

Isolation of Splenocytes, Lymph Node Cells, and CNS Mononuclear Cells

   Cells were isolated from mouse spleen and cervical lymph node by
   mashing tissues between two frosted microscope slides. The cells were
   further treated with RBC lysis buffer (Gibco, catalog number: A1049201)
   to eliminate erythrocytes, washed, and resuspended in RPMI 1640 (Gibco,
   catalog number: 31800022) supplemented with 10% FBS, 2 mM L-glutamine,
   100 U/ml penicillin G, 0.1 mg/ml streptomycin, and 10 mM HEPES (Life
   Technologies, Waltham, MA, USA). Isolation of CNS mononuclear cells was
   achieved using Percoll-gradient separation (GE Healthcare Bio-Sciences,
   Uppsala, Sweden) as previous described ([58]20).

Isolation of CD11b^+CD45^intTmem119^+ Microglia From CNS Mononuclear Cells

   Live CD11b^+CD45^intTmem119^+ microglia were isolated by cell sorting
   using a FACSAria Fusion (BD Biosciences, USA). After sorting, we
   sampled 300 cells (by the flow cytometry) for purity check to make sure
   the population is >95% microglia.

Hematoxylin and Eosin Stains and Immunofluorescence

   Mouse lumbar spinal cord sections were used for hematoxylin and eosin
   staining (H&E Staining Kit; Abcam, catalog number: ab245880), single
   myelin staining (FluoroMyelin Green Fluorescent, 1:300; Invitrogen,
   catalog number: [59]F34651), and triple-labeled immunofluorescence.
   Before primary antibody conjugation, additional blocking with
   mouse-on-mouse blocking reagents (Vector lab, catalog number:
   [60]R37621) was performed on each sample. 3-NT antibody (1:1,500;
   Abcam, catalog number: ab61392), in combination with antibody specific
   for CD11b (1:1,500; Bio-Rad, catalog number: MCA711G), ASPA (1:200;
   Millipore, catalog number: ABN1698), Neu-N (1:1,500; Abcam, catalog
   number: 177487), Iba-1 (1:1,500; WAKO, catalog number: 019-19741), or
   GFAP (1:1,500; DAKO, catalog number: 00087880), were used as indicated
   in the figure legends. Nox2 antibody (1:1,500; BD biosciences, catalog
   number: 611415) was counterstained with CD45 (1:1,500; BioLegend,
   catalog number: 103101), CD16/32 (1:1,500; BioLegend, catalog number:
   101301), Iba-1 or Neu-N. Phosphorylated p65 antibody (1:500; Millipore,
   catalog number: 3015337) was used to detect NF-κB activation in
   combination with CD11b and Neu-N. Spinal cord sections were then
   incubated with Alexa-488 (1:1,500; Invitrogen, catalog number: A32731),
   Alexa-568 (1:1,500; Invitrogen, catalog number: A-11011), and Alexa-647
   (1:1,500; Invitrogen, catalog number: A32733) conjugated secondary
   antibodies and mounted with DAPI (Vector lab, catalog number: 62248).
   The digital scanning analysis were kindly provided by the CMPB Image
   Analysis Lab at the NIEHS (LEICA-Scan scope FL System). All fluorescent
   images were obtained with a Zeiss LSM 780 laser scanning confocal
   microscope. Data were analyzed by Zen black version 2.0.

In Vivo Imaging System (IVIS)

   In vivo imaging was performed in mice after EAE induction to measure
   the levels of active myeloperoxidase (MPO) in activated phagocytes
   non-invasively. Before study, all the mice received hair removal at
   areas of interest to reduce the interreference of the desired signal.
   Anesthesia was induced with 2% isoflurane (Abbott Laboratories)
   inhalation in a special air tight transparent anesthesia box for 3–5
   min before the mice were moved to the light-tight chamber of the CCD
   camera in the imaging position. Bioluminescent images of inflammation
   at CNS area and MOG inoculation site were taken 10 min post
   intraperitoneal injection of the inflammation probe (XenoLight
   RediJect, PerkinElmer, 200 mg/kg) with IVIS Spectrum (PerkinElmer,
   5 min of exposure time). XenoLight RediJect Inflammation Probe is a
   ready-to-use chemiluminescent reagent and can be conveniently applied
   to study MPO activity of activated phagocytes. RediJect D-Luciferin
   (K^+ salt) is a bioluminescent in vivo substrate in a ready-to-use
   pre-formulated injectable format as a Luciferin-based conjugates as the
   bioluminescent imaging probe. The luminescence camera was set to 60 s
   exposure, medium binning, f/1, blocked excitation filter, and open
   emission filter. The photographic camera was set to 2 s exposure,
   medium binning, and f/8. Field of view was set to image all mice
   simultaneously. Identical settings were used to acquire each image and
   region of interest during the study as previously described. The
   luminescent areas of the CNS region and MOG inoculation site were
   defined as the region of interest (ROI) and the total signal in the ROI
   (photon/sec/m^2) was quantified using Living Image software 3D
   (version: 4.4.17197; PerkinElmer).

Flow Cytometry for T Cells and Myeloid Cells Analysis From Splenocytes,
Cervical Lymphoid Cells, and CNS Mononuclear Cells

   Before cytokine staining, T cells underwent in vitro stimulation with
   PMA (40 ng/ml), Ionomycin (2 μM), Monensin (4 μM) in 1 ml RPMI 1640
   (containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G, 0.1 mg/ml
   streptomycin, and 10 mM HEPES) for 4 h. Cells were resuspended in PBS
   supplemented with 0.5% BSA and 1 mM EDTA (FACS buffer) (splenocytes and
   lymphoid cells: 1 × 10^6/tube, all available CNS mononuclear cells from
   each sample/tube) and stained with the antibodies listed below at 4˚C
   for 30 min. The anti-mouse antibodies used for flow cytometric analysis
   were the following: anti–CD45–APC-Cy7, anti-CD4-APC, anti-IFNγ-BV421,
   anti-IL-17–PerCP Cy5.5, anti-IL-10–FITC, anti-Foxp3-PE,
   anti-CD11b-PE-Cy7, anti-Ly6G-BV421, anti-MHC II-FITC, anti-Ly6C-PerCP
   Cy5.5, anti-CCR2- BV510 (all antibodies were from eBiosciences or
   BioLegend). After staining, the cells were analyzed with FACSVerse flow
   cytometer. Flow cytometric data were viewed and analyzed by FlowJo v10.

RNA Extraction, Reverse Transcription, and Quantitative Real-Time PCR

   Homogenized mouse spinal cords (L4-spine level) or primary microglia
   culture were lysed with 1 ml TRIzol Reagent (Invitrogen). Nucleoprotein
   complexes were then separated through bromochloropropane
   (Sigma-Aldrich), and the samples were centrifuged at 12,000 rpm at 4˚C
   for 15 min. Total RNA was precipitated with an equal volume of
   isopropanol. The samples were incubated at 25˚C for 10 min and
   centrifuged at 12,000 rpm at 4˚C for 15 min. The RNA pellets were
   collected and washed with ethanol by vigorous mixing. RNA was obtained
   by centrifugation at 7,500 rpm at 4˚C for 10 min. After air-drying, the
   RNA pellets were dissolved in RNase-free double-distilled water.
   Reverse transcription was performed using a Reverse Transcription Kit
   (Applied Biosystems) to synthesize cDNA. Quantitative real-time PCR
   (qPCR) was performed using FastStart Universal SYBR Green Master with
   ROX (Roche), and cDNA was amplified using a StepOnePlus RealTime PCR
   System (Applied Biosystems). PCR cycling conditions were set as
   follows: 95°C for 10 min, then 40 cycles of 95°C for 15 s, 60°C for
   1 min, and 72°C for 20 s. The specificity of the SYBR green assay was
   confirmed by melting-point analysis. Expression data was calculated
   from the cycle threshold (Ct) value using the ΔCt method for
   quantification. Primer sequences are listed in [61]Supplementary Table
   1 .

Primary Cell Cultures

   Primary splenocytes culture for ELISA: 1 × 10^6/well, in 48-well plate,
   with or without MOG 10 μg/ml in 500 μl RPMI 1640 (10% FBS, 2 mM
   L-glutamine, 100 U/ml penicillin G, 0.1 mg/ml streptomycin, and 10 mM
   HEPES) for 72 h. Primary CNS mononuclear cells culture for ELISA: 1.5 ×
   10^5/well, in 48-well plate, with or without MOG 10 ug/ml in 500 μl
   DMEM/F12 (10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G, 0.1 mg/ml
   streptomycin, and 10 mM HEPES) for 72 h. Primary microglia culture
   (CD11b^+CD45^intTmem119^+) for RNA-sequencing and ELISA: 3 × 10^4/well,
   in 48-well plate, overnight seeding (18 h), then added MOG 10 μg/ml in
   500 μl DMEM/F12 (10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G, 0.1
   mg/ml streptomycin, and 10 mM HEPES) for 24 h.

Enzyme-Linked Immune Absorbent Spot (ELISpot)

   The frequency of IFNγ, IL-4, and IL-17–producing cells on preclinical
   (D7) and disease peak (D17) stage at the spleen were determined using
   ELISpot kit (Immunospot, C.T.L.) according to the manufacturer’s
   instructions. Splenocytes (5 × 10^5/well, in 200 ul) were plated in an
   ELISpot plate coated with rat anti-mouse IFNγ, IL-4, and IL-17 capture
   antibodies in the presence of 10 μg/ml MOG or PMA (2 mg/ml) plus
   Ionomycin (0.1 μg/ml) or no in vitro stimulation. Cells were cultured
   overnight and IFNγ, IL-4, and IL-17 production were detected using a
   biotin-conjugated rat anti-mouse IFNγ, IL-4, and IL-17 detection
   antibodies. Avidin-HRP was then added, and spots were developed with
   3-amino-9-ethylcarbamazole. Specific IFNγ, IL-4, and IL-17 production
   were calculated by the CTL ImmunoSpot^® Analyzers along with the
   ImmunoSpot^® software provided automated, objective recognition of
   spots, gating, and counting.

Multiplex Enzyme-Linked Immunosorbent Assay (ELISA)

   Plasma samples were analyzed by multiplex enzyme-linked immunosorbent
   assay (mouse cytokine/chemokine magnetic bead panel, MCYTOMAG-70K, EMD
   millipore). An array of 11 cytokines/chemokines was analyzed: GM-CSF,
   IFNγ, TNFα, IL-1β, IL-6, IL-10, IL-12p70, IL-17A, CCL2, CCL5, and
   CXCL10 using a Luminex—MAGPIX^® System and MILLIPLEX Analyst 5.1
   software. Assay Sensitivity: GM-CSF: 2.36 pg/ml; IFNγ: 1.04 pg/ml;
   TNFα: 3.04 pg/ml; IL-1β: 2.97 pg/ml; IL-6: 2.89 pg/ml; IL-10: 2.68
   pg/ml; IL-12p70: 3.40 pg/ml; IL-17A: 1.21 pg/ml; CCL2: 1.13 pg/ml;
   CCL5: 2.64 pg/ml; CXCL10: 2.20 pg/ml.

RNA-Seq Library Preparation and Sequencing

   Total RNA was subjected to cDNA synthesis and NGS (NextGen Sequencing)
   library construction using the Ovation SoLo RNA-Seq System (NuGEN
   Technologies, Redwood City, CA, USA). The quality and average length of
   sequence library for each sample was assessed using Bioanalyzer
   (Agilent Technologies, Santa Clara, CA, USA) and either the DNA 1000
   kit. The indexed samples were pooled equimolarily and sequenced on
   Illumina NovaSeq 6000 (150 bp, paired-end reads) (Illumina, San Diego,
   CA, USA).

Bioinformatics Analysis

   The quantification of raw RNAseq reads was processed using CLC Genomics
   Workbench v.10 software. Adaptor sequences and base with low quality or
   ambiguous were trimmed. The quality screened reads were mapped to mouse
   (GRCm38) genome using CLC Genomics Workbench. The mapping parameters
   were the following: mismatch cost 2, insertion cost 3, deletion cost 3,
   length fraction of 0.5, and similarity fraction of 0.8. The expression
   values were calculated as FPKM (Fragments Per Kilobases per Million).
   The differential gene expression between two or more condition based on
   the fold change of FPKM value. The genes with 2-fold change were
   further analyzed. The total transcripts from three samples (K-pooled,
   W1, and W2), 46,202, were filtered with protein-coding region first and
   left the items with either FPKM > 1 among these three samples. KEGG
   database ([62]21, [63]22) was used in pathway enrichment analysis and
   the pathway map was plotted by pathview ([64]23) package in R. Gene set
   enrichment analysis and Gene ontology enrichment analysis were done
   with the GSEA/MSigDB ([65]24) and GO-TermFinder ([66]25), respectively.
   Ingenuity Pathway Analysis (IPA, Qiagen, Germantown, MD, USA) was used
   to search for enriched canonical pathways.

Statistical Analysis

   Prism v5.03 software (GraphPad Software, San Diego, CA, USA) was used
   to generate graphs and for statistical analysis. Student unpaired t
   test was used for statistical analysis of the experiments in this
   study. All figures are presented as mean ± SEM. A p value 0.05 was
   defined as significant (*p < 0.05, **p < 0.01, ***p < 0.001; ns, not
   significant).

Results

Nox2 Deficiency Ameliorates EAE-Elicited Disease Severity, Body Weight Loss,
Leukocyte Infiltration, and Demyelination

   To investigate the role of Nox2 activity during EAE pathogenesis, we
   utilized three genotypes of female mice including Nox2 ^+/+ (W), Nox2
   ^+/− (H), Nox2 ^−/− (K) to determine the disease score and body weight
   changes ([67] Figures 1A, B ). The W and H mice showed similar
   time-related changes in clinical phenotype and symptoms onset beginning
   on 9~10 days post-injection (dpi) and then reached peak stage at 17
   dpi, while the K mice remained almost symptomless throughout the
   experiment. The severity of the EAE was evaluated by the degree of
   leukocyte infiltration (hematoxylin and eosin staining) and
   demyelination (fluoromyelin staining) in spinal cord sections (L4
   level) from each mouse strain. The dashed red lines outline the
   leukocyte invasion area and the dashed white lines mark the contour of
   demyelination location. These results showed that the W and H mice
   exhibited similar patterns of leukocyte infiltration and demyelination,
   whereas no clear lesions were observed in K mice ([68] Figures 1C, D ).
   In addition, the staining sections were evaluated blindly by a
   neuropathologist to give a quantitative score of infiltration and (0:
   normal, 5: most severe). Comparison of the EAE score and the pathologic
   score on disease peak (17 dpi) showed that these two scoring systems
   were well correlated ([69] Figure 1E ). In conclusion, Nox2-deficient
   mice show less inflammatory infiltration, demyelination at lumbar
   spinal cord after EAE induction on disease peak (17 dpi) compared with
   wildtype mice.

Figure 1.

   [70]Figure 1
   [71]Open in a new tab

   Nox2-deficient mice show less inflammatory infiltration, demyelination
   at lumbar spinal cord after EAE induction on disease peak (17 dpi). (A)
   EAE disease score and (B) Body weight change among three genotypes
   (mean ± SEM) [n = 33 (W), 63 (H), 46 (K)]. (C) Representative
   Hematoxylin and Eosin stains and (D) Fluoromyelin staining of lumbar
   spinal cord at the peak of EAE score (17 dpi). Left upper corner score:
   clinical score. Right upper corner score: pathologic score. The red dot
   lines outline the areas of leukocytes infiltration while the white dot
   lines outline the areas of demyelination. (E) Clinical score and
   pathologic score on day 17 based on the results of H&E stains [n = 7
   (W), 11 (H), 10 (K)]. Genotype: W, Nox2 ^+/+; H, Nox2 ^+/−; K, Nox2
   ^−/−. Formalin-fixed frozen section: 10 μm; spinal cord level: lumbar
   fourth, myelin: green; DAPI: blue. p value: *** < 0.001, * < 0.05; ns,
   not significant.

Nox2 Deficiency Greatly Reduces EAE-Elicited MPO Activity Within the CNS and
MOG Inoculation Areas

   For longitudinal tracking of phagocyte-mediated inflammatory status
   ([72]26), we injected the different mouse strains with the
   chemiluminescent XenoLight RediJect Inflammation Probe to measure MPO
   activity in activated phagocytes after EAE induction ([73] Figure 2A ).
   Bioluminescence here was used to assess the dynamic change of
   inflammation from CNS and MOG injection areas in vivo. Before being
   injected, mice were shaved at CNS and MOG inoculation areas for better
   signal detection ([74] Figure 2B ). Daily imaging analysis showed that
   the MPO signal present at the CNS area started to emerge at 8 dpi and
   was sustained up to 17 dpi in control animals, while almost no signal
   was detected in Nox2-deficent mice at these times ([75] Figures 2A ).
   Quantitative data on early (9 dpi) and peak disease (17 dpi) stage were
   shown on [76]Figures 2C, D . In summary, Nox2-deficient mice show
   greatly reduced MPO signal at both CNS and MOG injection areas compared
   with wildtype mice which implies less oxidative stress and
   neuroinflammation status from early through peak disease stages.

Figure 2.

   [77]Figure 2
   [78]Open in a new tab

   In vivo imaging analysis shows that EAE induction enhances MPO activity
   in both the CNS and MOG inoculation areas. (A) Serial daily imaging
   analysis showing the MPO signal at CNS and MOG inoculation areas. Right
   upper corner shows clinical EAE score. (B) Graph showing the shaved CNS
   and MOG inoculation areas for the detection of chemiluminescent signal
   by the IVIS Spectrum. (C) Early and (D) peak stages imaging: the MPO
   signal (mean ± SEM) from CNS and MOG inoculation areas on early disease
   stage (9 dpi) and peak stage (17 dpi) respectively. CNS area: day 9: n
   = 16 (W), 12 (K); day 17: 13 (W), 10 (K). MOG area: day 9: n = 9 (W), 5
   (K); day 17: 4 (W), 4 (K). Genotype: W, Nox2 ^+/+; K, Nox2 ^−/−. p
   value: ** < 0.01, * < 0.05.

Nox2 Deficiency Specifically Reduces the EAE-Elicited Invasion of Immune
Cells Into the CNS

   To determine the immune response and immune cells population among
   peripheral lymphoid organs and CNS, we evaluated the proliferation and
   activation of T cells, neutrophils, and monocytes in the spleen, the
   cervical draining lymph nodes, and the CNS of each mouse strain at 17
   dpi to study the distribution of pathogenic immune cells. No
   significant difference was observed between these mice strains
   regarding the levels of IFNγ (T[H]1)-producing T cells, IL-17A
   (T[H]17)-producing T cells, or IL-10- and Foxp3-producing regulatory
   CD4^+ T cells in their spleens or cervical draining lymph nodes ([79]
   Figure 3A ). In contrast, the number of these T cells present within
   the CNS was reduced in the Nox2-deficinet mice relative to controls
   ([80] Figure 4A ). We also were unable to detect differences in the
   number of neutrophils (Ly6G^+ MHC II^−), monocytes (Ly6G^−MHC II^+), or
   pathogenic monocytes (Ly6G^−MHCII^+ Ly6C^+CCR2^+) ([81]27) that
   infiltrated the spleen or cervical lymph nodes of the W and K mice
   ([82] Figure 3B ). However, the number of all these cell populations
   was greatly decreased in the CNS in Nox2-deficient mice ([83] Figures
   4B, C ). Consequently, the peripheral T cells and myeloid cells profile
   are similar at both groups, but great reduction was observed in
   Nox2-deficient mice in CNS on disease peak, which implies that
   peripheral immune cells are already boosted and activated, but retained
   in the periphery rather than entering CNS.

Figure 3.

   [84]Figure 3
   [85]Open in a new tab

   Nox2-deficient group presented similar profile of peripheral immune
   response as Nox2-competent group in spleen and cervical lymph nodes on
   disease peak (17 dpi). (A) the T cell profiles of IFNγ (T[H]1), IL-17A
   (T[H]17), IL-10, and Foxp3 (Treg)-producing cells from splenocytes
   (spleen) and cervical lymph nodes (CLN) respectively between W and K
   groups (n = 5 each group). (B) the myeloid cell profiles
   (CD11b^+CD45^hi) of neutrophils (Ly6G^+MHC II^−), monocytes (Ly6G^-MHC
   II^+), and cell count of pathogenic monocytes
   (Ly6G^-MHCII^+Ly6C^+CCR2^+) from spleen and CLN respectively between W
   and K groups (n = 5 each group). p value: ns, not significant.

Figure 4.

   [86]Figure 4
   [87]Open in a new tab

   Nox2 deficiency greatly reduces the EAE-elicited invasion of pathogenic
   immune cells into the CNS on disease peak (17 dpi). (A) the T cell
   profiles of IFNγ (T[H]1), IL-17A (T[H]17), IL-10, and Foxp3
   (Treg)-producing cells from CNS mononuclear cells between W and K
   groups (n = 5 each group). (B) the myeloid cell profiles
   (CD11b^+CD45^hi) of neutrophils (Ly6G^+ MHC II^−), monocytes (Ly6G^−MHC
   II^+), and (C) the absolute cell counts of pathogenic monocytes
   (Ly6G^−MHCII^+ Ly6C^+CCR2^+) from CNS mononuclear cells between W and K
   groups (n = 5 each group). p value: * < 0.05; ns, not significant.

Nox2 Deficiency Specifically Decreases the EAE-Elicited Increase in the
Levels of Cytokines and Chemokines Present Within the CNS

   To determine the cytokines/chemokines gene expression profile in the
   spinal cord and the protein levels from isolated CNS monocytes, we
   measured levels of cytokine and chemokine-encoding mRNA in the spinal
   cord (L4-level section) as well as the levels of cytokines and
   chemokines secreted from cultured monocyte isolated from the CNS at 17
   dpi. MOG treatment increased mRNA levels of cytokines Ifnγ (T[H]1) and
   Il-17a (T[H]17) as well as chemokines Ccl2, Ccl5, Ccl6, and Cxcl10 in W
   mice. However, Nox2 deficiency weakened these MOG-elicited immune
   responses ([88] Figure 5A and [89]Supplementary Figure 1A ). By
   contrast, the levels of Il-4 and Ccl20 mRNAs manifested minimum
   differences between the W and K MOG-injected mice ([90] Supplementary
   Figure 1A ). We also utilized ELISA to quantify the levels of cytokines
   (GM-CSF, IFNγ, TNFα, IL-1β, IL-6, IL-10, IL-12p70, and IL-17A) and
   chemokines (CCL2, CCL5, CXCL10) secreted from primary CNS-isolated
   mononuclear cells at 17 dpi ([91] Figure 5B and [92]Supplementary
   Figure 1B ). To enhance the production of cytokines and chemokines from
   these cells, MOG (10 μg/ml) was added to their culture media for 3
   days. MOG treatment caused a large increase in the supernatant levels
   of GM-CSF, IFNγ, TNFα, IL-6, IL-10, IL-17A, CCL2, CCL5, and CXCL10
   produced by CNS monocytes isolated from W mice, but not those isolated
   from K mice. In contrast, no difference was observed in the levels of
   IL-1β or IL-12p70 secreted by CNS monocytes isolated from these mouse
   strains ([93] Figure 5B and [94]Supplementary Figure 1B ).

Figure 5.

   Figure 5
   [95]Open in a new tab

   Nox2 deficiency diminishes EAE-elicited increase in mRNA expression in
   the spinal cord and secreted proteins of cytokine and chemokines from
   CNS mononuclear cells on disease peak (17 dpi). (A) mRNA levels (mean ±
   SEM) of spinal cord (L4-level section, 0.5 μm thick tissues) at 17 dpi
   after MOG inoculation were determined by RT-PCR. Cytokines: Ifnγ, Il-4,
   Il-17a; chemokines: Ccl2, Ccl5, Cxcl10. All data were normalized as
   fold of Naïve-W group. (n = 3 to 5 for each group) (B) The amount of
   cytokines (GM-CSF, IFNγ, IL-17A) and chemokines (Ccl2, Ccl5, Cxcl10)
   secreted from primary mononuclear cells isolated from the CNS collected
   at 17 dpi after MOG inoculation were quantified by ELISA assays. (n = 3
   to 5 for each group). Cell culture condition was described in the
   results. NS, no drug treatment; MOG: 10 μg/ml. p value: *** < 0.001, **
   < 0.01, * < 0.05, ns, not significant.

   To investigate the immune response in the spleen of our MOG-injected
   mice, we first used the ELISpot assay to quantify the levels of IFNγ,
   IL-4, and IL-17A secreted from total splenocytes at 7 dpi (before any
   clinical presentation) and 17 dpi (disease peak). The W and K groups
   showed no significance was found between Nox2-deficent and wildtype
   control groups ([96] Supplementary Figure 2A ). We then used an ELISA
   assay to quantify the amount of cytokines (GM-CSF, IFNγ, TNFα, IL-1β,
   IL-6, IL-10, IL-12p70, and IL-17A) and chemokines (CCL2, CCL5, and
   CXCL10) secreted from these splenocytes at 17 dpi. However, we were
   unable to detect any difference in levels of cytokines and chemokines
   secreted by splenocytes isolated from our mouse strains ([97]
   Supplementary Figure 2B ).

   Collectively, these gene expression and protein level of cytokines and
   chemokines profiles in spleen are similar in both groups, while great
   reduction was found in Nox2-deficient mice in CNS. Therefore, not only
   the number of autoreactive T cells and myeloid cells showed great
   reduction in Nox2-deficient mice, but also similar trend was found in
   the cytokines and chemokines production in CNS on disease peak.

Nox2 Deficiency Largely Reduces the Levels of Oxidative Stress in EAE Mice

   To determine the effect of Nox2-deficincy on the levels of oxidative
   stress experienced in EAE mice in the CNS, we examined levels of
   3-Nitrotyrosine (3-NT), the leukocyte marker CD45, and the
   microglia/macrophage marker Iba-1 in MOG-injected W and K mice over a
   period of 6 weeks ([98] Figure 6A and [99]Supplementary Figure 3A ) in
   the spinal cord (L4 level). The W mice showed an abrupt increase of all
   these markers at 14 dpi, while the K mice showed little changes in the
   levels of these markers at this time. The 3-NT signal lasted for 2
   weeks and subsided after 28 dpi, while the immunoreactivity of CD45 and
   Iba-1 remained elevated up to 42 dpi in W mice. It is important to note
   that MOG inoculation greatly increased the mRNA expression of two key
   superoxide free radical-producing molecules, Nox2 and CD11b (a-chain of
   Mac-1 receptor), in W mice from the spinal cord tissue ([100] Figure 6B
   ).

Figure 6.

   [101]Figure 6
   [102]Open in a new tab

   Nox2 deficiency largely reduces the production of oxidative stress and
   the signal of pNF-κB on disease peak (17 dpi) at spinal cord area. (A)
   Time-dependent of each parameter in lines as a quantitative manner. The
   mean fluorescence intensity of whole pixels was measured by Image J.
   (B) mRNA levels (mean ± SEM) of spinal cord (L4-level section, 0.5 μm
   thick tissues) at 17 dpi after MOG inoculation were determined by
   RT-PCR. Cell membrane protein: CD11b and Nox2, (C) The relationship
   between 3-NT (3-Nitrotyrosine, oxidative stress marker) signal and
   Iba-1 (microglia/macrophage), CD45 (leukocytes), CD11b
   (microglia/macrophage), (D) ASPA (aspartoacylase, active
   oligodendrocytes), (E) GFAP (astrocytes), (F) NeuN (total neurons). (G)
   The relationship between pNF-κB (inducible inflammatory transcription
   factor) and NeuN, CD11b. Genotype: W, Nox2 ^+/+; K, Nox2 ^−/−.
   Formalin-fixed frozen section: 10 μm. Spinal cord level: lumbar fourth.
   (C–F) marginal and ventral area of spinal cord (white matter); (G)
   ventral area of spinal cord (gray matter). p value: *** < 0.001, ** <
   0.01, * < 0.05, ns, not significant.

   To determine cell type responsible for increasing the oxidative stress
   levels in EAE mice, we performed doubling-immunostaining of 3-NT and
   markers for different cell types: Iba-1 and CD11b for
   microglia/macrophage, CD45 for leukocytes, aspartoacylase (ASPA) for
   active mature oligodendrocytes, GFAP for astrocytes, and NeuN for
   neurons ([103] Figures 6C–F ). In MOG-treated W mice, high
   immunoreactivity of 3-NT was observed in cells expressing CD45, CD11b,
   and Iba-1; to a lesser degree in NeuN-positive cells, but not in either
   ASPA- or GFAP-positive cells. In contrast, little detectible 3-NT
   immunoreactivity was found in all the cell types mentioned above in
   MOG-treated K mice. Mean fluorescent intensity of immunofluorescent
   slides: 3-NT (W: 10.16 ± 0.51, K: 5.24 ± 0.21, p < 0.001), CD45 (W:
   9.80 ± 0.66, K: 1.81 ± 0.33, p < 0.001), CD11b (W: 12.58 ± 1.11, K:
   3.74 ± 0.34, p < 0.01), Iba-1 (W: 10.29 ± 1.10, K: 1.76 ± 0.59, p <
   0.01), NeuN (W: 8.78 ± 1.26, K: 10.14 ± 0.74, p: not significant), ASPA
   (W: 7.70 ± 0.52, K: 9.40 ± 0.25, p < 0.05), and GFAP (W: 10.44 ± 0.58,
   K: 5.392 ± 0.65, p < 0.01).

   Regarding the costaining between Nox2 level and Iba-1, CD45, CD11b,
   CD16/32 (proinflammatory marker of microglia/macrophage/granulocytes)
   ([104]28), and NeuN (a neuronal marker) at 17 dpi ([105] Supplementary
   Figures 3B, C ), Nox2 signal was mostly detected in CD45, CD11b, and
   Iba-1 positive cells, while it was almost undetectable in NeuN positive
   cells. Moreover, CD16/32, M1 activation marker, was abundant at CD45
   population, which implies the presence of an active inflammatory
   response at the peak of disease. Mean fluorescent intensity of
   immunofluorescent slides: Nox2 (W: 12.15 ± 1.02, K: 1.54 ± 0.63, p <
   0.001), CD16/32 (W: 8.31 ± 0.55, K: 1.68 ± 0.37, p < 0.001).

   Since ROS activates NF-κB signaling, which modulates gene expression in
   diverse cellular processes such as innate immune response ([106]29,
   [107]30), we next determined if NF-κB plays were required for the
   expression of inflammatory transcription factors in our EAE model. To
   do this, we co-stained the active form of NF-κB (pNF-κB) with NeuN or
   CD11b ([108] Figure 6G ). The W mice exhibited an abundant pNF-κB
   signal, which partially colocalized with CD11b, but not with NeuN. Yet,
   the K mice showed almost no pNF-κB signal at 17 dpi. Mean fluorescent
   intensity of immunofluorescent slides: pNF-κB (W: 11.87 ± 1.06, K: 3.62
   ± 0.22, p < 0.01).

Nox2 Deficiency Diminishes the EAE-Elicited Increase in Microglia Activation
as Well as Cytokine/Chemokine Secretion in the CNS

   To investigate the specific contribution of microglia at this model, we
   utilized flowcytometry analysis showed that a 58.8% decrease in the
   number of microglia (CD11b^+CD45^int) ([109]20, [110]31) taken at 17
   dpi in K mice, as compared to controls ([111] Figure 7A ). Sorted
   microglia (CD11b^+CD45^intTmem119^+, purity >95%) ([112]32) from each
   group were then seeded in a density of 3 × 10^4/500μl/well. Following
   42 h incubation, supernatants from primary microglia culture were
   collected for ELISA analysis ([113] Supplementary Figure 4 ). The data
   show that levels of IL-6, IL-10, and CCL5 were lower in the
   MOG-injected K mice relative the MOG-injected W mice ([114] Figure 7B
   ). However, levels of GM-CSF, IFNγ, TNFα, IL-17A, CCL2, and CXCL10
   showed a trend of decrease in the K group compared to the W group.
   Nevertheless, these differences were not statistically significant.
   likely due to the small sample sizes. No difference was found for the
   levels of IL-1β and IL-12p70 between MOG-injected W and K mice ([115]
   Figure 7B ). Thus, Nox2-deficient microglia failed to secret enough
   cytokines and chemokines to induce the invasion of peripheral
   pathogenic immune cells into the CNS.

Figure 7.

   [116]Figure 7
   [117]Open in a new tab

   Cytokine/chemokine and transcriptome profiles of Nox2-deficient
   microglia. CD11b^+CD45^intTmem119^+ microglia were isolated from the
   CNS tissue of Nox2-competent and deficient mice on disease peak (17
   dpi). (A) microglia (CD11b^+CD45^int) count analyzed by flow cytometry
   (n = 5 each group). (B) Concentrations of cytokines and chemokines
   released from primary cultured microglia. [n = 4 (W), 2 (K-pooled)].
   MOG: 10 μg/ml. p value: * < 0.05; ns, not significant. (C) Relative
   mRNA levels of cytokine/chemokine genes in isolated microglia as
   determined by RNAseq. Color scales depict relative fold changes. (D)
   Significantly enriched KEGG pathways in association with immune
   response, inflammation, infection, and known EAE related pathways. (E)
   Correlations of the IFNγ-related pathway with Nox2-competent and
   deficient microglia transcriptome GSEA. p value: * < 0.05; ns, not
   significant.

Nox2 Deficiency Affects Multiple Cytokine/Chemokine- and Inflammation-Related
Pathways in Microglia

   Next, we performed RNAseq to determine the global impact of Nox2
   deficiency on microglia functions and to identify Nox2 associated
   pathways. Genome-wide transcriptome profiling identified 1621
   differentially expressed genes (DEG) with ≥ 3-fold changes between W
   and K microglia cells treated with exogenous MOG ([118] Supplementary
   Figure 4 and [119]Supplementary Table 2 ). In addition to the
   aforementioned changes in secreted cytokines and chemokines ([120]
   Figure 7B ), expression of chemotactic gene Pf4 ([121]33) and
   proinflammatory cytokines and chemokines Tnfrsf9, Tnfsf12, Tnfsf13,
   Ccl7, Cxcl3, and Cxcl9 ([122]34–[123]39) also showed a reduction in
   response to Nox2 deficiency ([124] Figure 7C ). These results indicate
   that Nox2 plays a broad role in regulating the activation of microglia
   ([125]1, [126]40). Gene ontology analysis performed on the KEGG
   pathways ([127] Supplementary Table 3 ) revealed DEG enrichment in six
   pathways that are associated with virus infection (human papillomavirus
   infection, herpes simplex virus 1 infection, and human T-cell leukemia
   virus 1 infection), which are known to be associated with EAE-related
   signaling pathways, such as the MAPK and PI3K-Akt signaling pathways as
   well as cytokine-cytokine receptor interaction ([128] Figure 7D ).
   Ingenuity Pathway Analysis further identified an enrichment of the
   STAT3 pathway ([129]41), glutathione-mediated detoxification ([130]42),
   leukotriene biosynthesis ([131]43), IL-8 signaling ([132]44), HMGB1
   signaling ([133]45), NRF2-mediated oxidative stress response ([134]46),
   systemic lupus erythematosus in B cell signaling pathway ([135]47), and
   T cell exhaustion signaling pathway ([136]48) in the Nox2 gene
   signature and are in association with MS or EAE ([137] Table 1 and
   [138]Supplementary Table 4 ). Importantly, Gene Set Enrichment Analysis
   (GSEA) found that 35 out of 42 significantly enriched gene sets from
   Nox2-competent microglia are inflammation or infection-related.
   However, none of the 13 gene sets identified in Nox2-deficient
   microglia belong to these categories ([139] Supplementary Table 5 ), as
   exemplified by the IFNγ-related pathway ([140] Figure 7E ).
   Collectively, our findings suggest that Nox2 is required for the
   activation of and cytokine/chemokine secretion by microglia, which has
   important implications on the neuroinflammation process in EAE.

Table 1.

   Nox2 dependent pathways in microglia with an association with multiple
   sclerosis or experimental autoimmune encephalomyelitis (EAE).
   Pathway p value (-log10) References