Yang et al. Journal of Neuroinflammation(2024) 21:181
https://doi.org/10.1186/s12974-024-03172-x

Journal of Neuroinflammation

Open Access

RESEARCH

2-Bromo-1,4-Naphthalenedione promotes
CD8+ T cell expansion and limits Th1/Th17
to mitigate experimental autoimmune
encephalomyelitis
Cuixia Yang1,3†, Yuanchen Ma2†, Qiying Lu3†, Yuliang Qu4, Yuantao Li5, Shimei Cheng3, Chongjun Xiao3,
Jinshuo Chen3, Chuangjia Wang3, Feng Wang7, Andy Peng Xiang5,6, Weijun Huang5*, Xiaorong Tang1* andHaiqing Zheng3*
Abstract
Treating Multiple sclerosis (MS), a well-known immune-mediated disease characterized by axonal demyelination,is challenging due to its complex causes. Naphthalenedione, present in numerous plants, is being exploredas a potential medicine for MS due to its immunomodulatory properties. However, its effects on lymphocytes
can vary depending on factors such as the specific compound, concentration, and experimental conditions. In
this study, we aim to explore the therapeutic potential of 2-bromo-1,4-naphthalenedione (BrQ), a derivative of
naphthalenedione, in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and to elucidate
its underlying mechanisms. We observed that mice treated with BrQ exhibited reduced severity of EAE symptoms,
including lower clinical scores, decreased leukocyte infiltration, and less extensive demyelination in central nervous
system. Furthermore, it was noted that BrQ does not directly affect the remyelination process. Through cell-chatanalysis based on bulk RNA-seq data, coupled with validation of flow analysis, we discovered that BrQ significantly
promotes the expansion of CD8+ T cells and their interactions with other immune cells in peripheral immune
system in EAE mice. Subsequent CD8+ T cell depletion experiments confirmed that BrQ alleviates EAE in a CD8+
T cell-dependent manner. Mechanistically, expanded CD8+ cells were found to selectively reduce antigen-specificCD4+ cells and subsequently inhibit Th1 and Th17 cell development in vivo, ultimately leading to relief from EAE.

†

Cuixia Yang, Yuanchen Ma and Qiying Lu contributed equally to thiswork.
Correspondence and requests for materials should be addressed to
Haiqing Zheng.
*Correspondence:
Weijun Huang
hweijun@mail.sysu.edu.cn
Xiaorong Tang
372730132@qq.com
Haiqing Zheng
zhenghq2@mail.sysu.edu.cn
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Yang et al. Journal of Neuroinflammation(2024) 21:181

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In summary, our findings highlight the crucial role of BrQ in modulating the pathogenesis of MS, suggesting its
potential as a novel drug candidate for treating MS and other autoimmune diseases.
Keywords 2-bromo-1,4-Naphthalenedione, CD8+ T cells, Experimental autoimmune encephalomyelitis, Th1, Th17

Introduction
Multiple sclerosis (MS) is the predominant chronic autoimmune disorder that affects the central nervous system(CNS), with a global impact on affecting nearly 3 million
individuals. Typically appearing between the ages of 20
and 40, MS is the primary nontraumatic cause of neurologic disability among young adults. It disproportionately
affects women, with a ratio of approximately two to one[1, 2]. Common symptoms of MS include limb weakness
or sensory loss, ataxia, visual disturbances, spasticity,
fatigue, pain, and cognitive deficits. Widely recognized
today, MS is believed to stem from a complex interplayof multiple genetic, epigenetic, and environmental risk
factors. Its pathogenesis unfolds through a series of pathological processes, beginning with immune system activation, followed by the infiltration of immune cells acrossthe blood–brain barrier (BBB), demyelination, gliosis,
and neuroaxonal degeneration. These cascading events
culminate in the disruption of neuronal signaling, giving
rise to neurologic syndromes and physical disability [3,4].
Presently, clinical interventions primarily target the
peripheral inflammatory processes that initiate during
the early stages of MS. This treatment spectrum includes
a variety of medications such as beta-interferons, teriflunomide, glatiramer acetate, dimethyl fumarate, mitoxantrone, ocrelizumab, and alemtuzumab. Recognizing thecharacteristic immune dysregulation observed in MS,
which prompts the infiltration of autoreactive immune
cells into the CNS and precipitates neurodegeneration,
supplementary medications like fingolimod and natalizumab have been developed to further curb immune cell
infiltration [5–7]. Although these therapies have demonstrated efficacy in alleviating disease severity, they only
partially impede MS progression, falling short of achieving full remission. Furthermore, due to the lack of specificity in current treatments, the more potent medicationsfor MS often entail a heightened risk of serious adverse
events. For instance, adverse events associated with oral
fingolimod in relapsing multiple sclerosis include bradycardia, atrioventricular conduction block, elevatedliver enzyme levels, macular edema, and mild hypertension [8]. Similarly, natalizumab, along with other immunotherapeutic drugs, is linked to progressive multifocal
leukoencephalopathy [7, 9]. Consequently, the therapeutic landscape for multiple sclerosis remains daunting,emphasizing the urgent need for the development of
new medications with promising efficacy and fewer side
effects.

For centuries, Traditional Chinese Medicines havebeen utilized in China and Europe to address a varietyof illnesses. Fascinatingly, many of these medicines contain quinones, which possess diverse biological functions, including combating viruses, bacteria, fungi,
malaria, cancer, and Alzheimer’s disease [10, 11]. Naphthoquinones, a subtype of quinone, have garnered significant attention in immunology and related fields.
Research has demonstrated that 1,4-Naphthoquinone
targets P2 × 7 purinergic receptors for anti-inflammatory
effects [12, 13]. Lapachol reduces lymphocyte proliferation and severity of autoimmune arthritis [14]. Juglone,from walnut husks, has anti-diabetic, anti-hypertensive,
anti-tumor properties, and improves ulcerative colitis[15]. Plumbagin, derived from medicinal plants, has beenshown to ameliorate experimental autoimmune encephalomyelitis [16]. Shikonin in Lithospermum erythrorhizon
is effective against various autoimmune diseases [17].
Vitamin K3 (menaquinone) may aid in the treatment
of NLRP3-related inflammatory conditions [18]. Thesefindings underscore the immunosuppressive effects ofnaphthoquinones, although their effects on lymphocytes
can vary depending on factors such as the specific compound, concentration, and experimental conditions.
The experimental autoimmune encephalomyelitis(EAE) mouse model, which closely mimics many clinical,
pathological, and histological features of MS, is widely
utilized in MS drug discovery. We assessed the effects of
1,2-naphthoquinone, 1,4-naphthoquinone, 2-naphthol,
menadione, 2,3-dichloro-1,4-naphthoquinone, quinoclamine, 2-chloro-1,4-naphthoquinone, 2-bromo-1,4-naphthoquinone, and 2-methoxy-1,4-naphthoquinone on
EAE. The results demonstrated that BrQ exhibited themost significant and safe effect in alleviating EAE clinical symptoms, making it the most promising candidate
for further investigation in MS treatment. In this study,
the clinical scores, H&E and Luxol fast blue staining
were used to determine the protective effect of BrQ on
EAE mice. Oligodendrocyte precursor cells (OPCs) differentiation assay and cuprizone induced demyelination
model were performed to evaluate the direct effect of
BrQ on remyelination processes in vitro and in vivo,
respectively. Further bulk RNA-seq, cell-chat analysis
and flow analysis validation were conducted to uncoverintercellular communication networks, as well as potential mechanisms and therapeutic targets. Overall, weshow for the first time that BrQ significantly alleviates
the severity of EAE mice and could be developed as a

Yang et al. Journal of Neuroinflammation(2024) 21:181

unique therapeutic agent for the treatment of MS andother autoimmune diseases.

Materials and methods
Mice

C57BL/6 mice were purchased from Beijing Vital River
Laboratory Animal Technology (Beijing, China). All
mice were maintained in pathogen-free conditions withstandard laboratory chow and water ad libitum in the
Sun Yat-sen University animal care facility. Sex- and agematched littermates between 8 and 10 weeks of age wereused for animal experiments. All animal experiments
were performed with the approval of Sun Yat-sen University Institutional Animal Care and Use Committee.
Induction and treatment of EAE

The EAE animal model was developed as previously
described [19]. In brief, male C57BL/6 mice were immunized subcutaneously with 200 µg MOG35 − 55 peptide(MEVGWYRSPFSRVVHLYRNGK, GL Biochem, Shanghai, China) emulsified in Complete Freund’s adjuvant,which contains 5 mg/ml heat-killed Mycobacterium
tuberculosis (H37Ra strain, BD Diagnostics). Each mousereceived a total volume of 200 µl of MOG35 − 55 peptide/
CFA emulsion, with 50 µl injected subcutaneously at two
separate sites on the back, resulting in four injections per
mouse. Pertussis toxin (dissolved in sterile distilled water,
200 ng/mouse, List Biological Labs) was injected intraperitoneally on day 0 and day 2 following immunization.
The mice were monitored daily and clinical appearance was scored on a scale of 0–5 as follows: 0, no clinicalsigns; 1, paralyzed tail; 2, paresis (weakness, incomplete
paralysis of one or two hindlimbs); 3, totally paralyzed
hind legs; 4, paraplegia with weak or paralyzed forelimbs;
5, death. Add or subtract 0.5 points if the disease severity
is between two scales.
In the prophylactic treatment regimen, EAE mice weredivided into four groups: vehicle, BrQ low dose group(3 mg/kg), BrQ medium dose group (10 mg/kg) and BrQhigh dose group (30 mg/kg). 2-bromo-1,4-naphthalenedione (abbreviated as BrQ, Sigma, CAS: 2065-37-4,
510,300) was dissolved by sonication in a 0.5% carboxymethylcellulose sodium solution (CMC) suspension andaliquoted for freezing at -20 °C, with one vial used per
extraction without repeated freeze-thaw cycles. The mice
were orally administered at 9:00 am every day from day 3
post-immunization until the end of the study. The vehicle
group was given an equal volume of 0.5% CMC (200 µL
per mouse) by gavage. For the treatment regimen, theEAE mice were orally gavaged at a dosage of 30 mg/kg
daily starting on day 7, day 14, or day 21 after immunization until the end of the study.

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Histopathological and immunohistochemical analysis

For the histological analysis, vehicle and BrQ-treated
EAE mice were deeply anesthetized with 1.25% tribromoethanol (15 µL/g body weight) on day 18 post-immunization and then transcardially perfused with 20 ml 0.9%
NaCl solution to remove peripheral blood from internalorgans followed by 15 ml 4% (w/v) paraformaldehyde(pH 7.2). The lumbar spinal cord was carefully collected
and fixed in 4% (w/v) paraformaldehyde overnight at 4℃.
Paraffin-embedded sections were prepared. 5-µm-thicktransverse paraffin sections were stained with Hematoxylin and Eosin (H&E) or Luxol fast blue (LFB, Sigma,
S3382) for visualizing the number of infiltrating cells andmyelin loss in inflammatory foci per section, respectively.
Image-Pro software was utilized for quantifying levels of
inflammation and demyelination, respectively.
Isolation and analysis of CNS leukocyte infiltration

Brain and spinal cord isolated from 0.5% CMC and BrQtreated EAE mice after perfusion with 0.9% NaCl solution on day 18 post-immunization were homogenizedin ice-cold tissue grinders. Single-cell suspensions wereobtained by filtering through 70-µm cell strainers and thecells were collected by centrifugation at 500×g for 10 min
at 4 °C. The cells were resuspended in 8 ml 37% isotonic
Percoll (GE Healthcare, USA) and overlaid onto 4 ml 70%
isotonic Percoll cushion in 15 ml tubes. The gradient wascentrifuged at 780×g for 20–30 min at 25 °C. Cells collected at 37/70% (vol/vol) Percoll interface were re-stimulated for 4–6 h at 37 °C in vitro and subjected to flow
cytometry with cell surface, intracellular characteristic
cytokine, or transcription factors staining. According to
the manufacturer’s instructions, CountBright™ absolutecounting beads (Invitrogen, C36950) were used to quantify the absolute number of total CNS leukocyte infiltration, as well as CNS infiltrating CD4+ T cells, including
Th1, Th17 and Treg cells.
CD4+ T cell isolation and in vitro Th1/Th17/Treg
differentiation

Naive CD4+ T cells were prepared from the spleen of
C57BL/6 mice (7–9 weeks of age) by magnetic depletion
separation (BioLegend, 480040). Naive CD4+ T cells were
cultured in complete RPMI 1640 containing 10% FBS,
2-mercaptoethanol (50 µM), L-glutamine (2 mM), 100
Units/mL penicillin and 100 µg/ml streptomycin. The
cells were activated with anti-CD3 (2 µg/ml, BioLegend,
100331) and anti-CD28 (2 µg/ml, BioLegend, 102112).
Anti-IL-4 (BioLegend, 504115, 10 µg/ml) and IL-12 (10ng/ml) were added for Th1 polarization. For Th17 differentiation, the cells received anti-IL-4 (10 µg/ml) andanti-IFN-γ (BioLegend, 505810, 10 µg/ml) plus a Th17
mixture containing IL-6 (30 ng/ml), TGF-β1 (3 ng/ml),
IL-23(10 ng/ml), TNF (10 ng/ml) and IL-1β (10 ng/ml).

Yang et al. Journal of Neuroinflammation(2024) 21:181

Polarization of Treg was induced by the addition of IL-2
(10 ng/ml), TGF-β1 (5 ng/ml) and anti-IFN-γ (10 µg/ml).
BrQ at various concentrations was added to assess theirinfluence on Th1, Th17, and Treg cell differentiation invitro. Cells were collected for analysis after incubation for
3–4 days.
Flow cytometry

CNS infiltrates, draining lymph node cells or CD4+ T
cells from differentiation assays in vitro were stimulated with PMA (50 ng/ml, Sigma), ionomycin (750 ng/
ml, Sigma), and brefeldin A (10 µg/ml, TargetMol) for
4–6 h at 37 °C. Surface markers (CD4, CD8, B220, andCD11b) were stained for 30 min in the dark at 4 °C withthe appropriate antibodies, then the cells were washedand resuspended with 1xPBS. For intracellular cytokine
IL-17 A and IFN-γ analysis, cells were fixed and permeabilized with an Intracellular Fixation & Permeabilization
Buffer Set (eBioscience, 88-8824) according to the manufacturer’s protocol following surface staining. For Foxp3
staining, a Foxp3 staining buffer set (eBioscience) wasused following the manufacturer’s instructions.
Cells were analyzed with CytoFLEX S System (Beckman Coulter). Flow cytometry data were plotted andquantified with FlowJo 10.8.1 software. The following
antibodies were used: Pacific Blue™ anti-mouse CD4antibody (Biolegend, 100531, 1:100), PE/Cyanine7 antimouse CD8a antibody (Biolegend, 100722, 1:100), APC
anti-mouse/human CD45R/B220 antibody (Biolegend,
103212, 1:100), FITC anti-mouse/human CD11b antibody (Biolegend, 101206, 1:100), PE anti-mouse IL17a(Biolegend, 506904, 1:100), APC anti-mouse IFN-γ (Biolegend, 505810, 1:100), FITC anti-mouse Foxp3 (eBioscience, 11-5773-82, 1:100). Flow cytometry analysis was
conducted by CytoFLEX flow cytometer (Beckman Coulter) and assessed with the FlowJo 10.8 software.
Cytokine cytometric bead array (CBA) assay

The whole blood was collected from EAE mice treatedwith either 0.5% CMC solution or 30 mg/kg BrQ (administered orally, starting from day 3) on day 10 postimmunization. After collection, the blood samples wereallowed to clot at room temperature for 30 min, followedby centrifugation at 1,000 g for 10 min at 4˚C to obtainthe serum. Subsequently, the prepared supernatant wastransferred to RNase/DNase-free tubes, aliquoted andstored at -80 °C until further analysis. The concentrationsof IFN-γ, IL-17a, TNF, IL-10, and IL-6 in the serum were
quantified using the Cytometric Bead Array Mouse Cytokine Kit (BD Biosciences, 560,485) according to manufacturer instructions. Cytometric bead array data wereacquired by CytoFLEX flow cytometer (Beckman Coulter) using CytExpert software and analyzed using BD
Cytometric Bead Array software (BD Biosciences).

Page 4 of 18

In vivo CD8+ T cells depletion

The EAE mice were randomly assigned to four groups
in a blinded fashion: Isotype + Vehicle; Isotype + 30 mg/
kg BrQ; anti-CD8 antibody + Vehicle; anti-CD8 antibody + 30 mg/kg BrQ (n = 6–8 per group). The anti-CD8
antibody (BioXcell, BE0004-1, Clone:53 − 6.7) was intraperitoneally administered to EAE mice at a dose of 100 µg
on both day 3 and day 4 following immunization. Subsequently, a supplementary dose of 30 µg anti-CD8 antibody was administered once on day 11. Control animals
received an equal dose of isotype antibody (BioXcell,
BE0089, Clone:2A3). Careful attention was given to dosage and timing to ensure effective depletion of CD8+ T
cells in EAE mice while maintaining mouse survival rates.
The efficacy of the depletion was assessed through FACS
analysis of peripheral blood T cells on days 10 and 15,
respectively. The results revealed a clearance efficiencyof 71.2% on day 10 and 58.3% on day 15 (Supplementary
Fig. 3). Following CD8+ T cell depletion or BrQ treatment, the progression of EAE symptoms was observed toevaluate the contribution of CD8+ T cells in BrQ-mediated amelioration of EAE clinical disease severity.
CFSE-based proliferation assays

Total leukocytes were obtained from the draining lymph
nodes of 8-9-week-old C57BL/6 mice, primarily collectedfrom the axillary, lateral axillary, and superficial inguinal regions. Naive CD8+ T cells were isolated using the
MojoSort™ Mouse CD8+ T Cell Isolation Kit (Biolegend,
480035). The leukocytes and naive CD8+ T cells werethen adjusted to a cell concentration of 1 × 106 cells/mL
with pre-warmed PBS solution. Next, an appropriate
volume of CellTrace™ CFSE stock solution (Invitrogen,
C34554, diluted at 1:2000) was added to the cell suspension to achieve a final working solution of 2.5 μM. Subsequently, the cells were incubated for 20 min at 37 °C
in a water bath, protected from light. Following two
washes, the cells were cultured in complete RPMI 1640
medium supplemented with 10% FBS, 2-mercaptoethanol (50 µM), L-glutamine (2 mM), 100 Units/mL penicillin, and 100 µg/ml streptomycin, anti-CD28 (5 µg/ml),
anti-CD3 (5 µg/ml), and IL-2 (20 ng/ml). After 3 days ofculture, CD8 surface staining was performed and CFSElabeled cells were analyzed using flow cytometry to assess
proliferation within the CD8+ gate.
Bulk RNA-seq and data analysis

The lymphocytes in the dLN on day 10 after immunization were obtained from EAE mice treated with 0.5%
CMC solution or 30 mg/kg BrQ (each group contains
two pools, n = 3 mice per pool). After washing twice with
1xPBS, the cells were harvested for total RNA extraction by Trizol reagent (Invitrogen, 15596-018) following the manufacturer’s procedure. Then, the samples

Yang et al. Journal of Neuroinflammation

Fig. 1 (See legend on next page.)(2024) 21:181

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Yang et al. Journal of Neuroinflammation(2024) 21:181

Page 6 of 18(See figure on previous page.)
Fig. 1 BrQ administration attenuates clinical symptoms of EAE mice. A Chemical structure of BrQ. B Schematic diagram of MOG35 − 55-induced EAE modeland BrQ treatment. C Clinical scores of EAE mice treated with vehicle (0.5% CMC) or different dosages of BrQ (3, 10, or 30 mg/kg body weight) daily by
gavage starting from day 3 after immunization, n = 5–6 mice per group. D The stacked column depicts EAE clinical incidence and severity from day 9 to
day 21. E, G H&E staining and LFB staining of paraffin-embedded sections of spinal cords isolated from vehicle- or BrQ-treated EAE mice on day 18 postimmunization. Scale bars: 400 μm. F, H Statistical analysis of CNS inflammatory cell infiltration in H&E-stained sections and demyelination areas in luxol
fast blue-stained spinal cord sections by Image-Pro software. n = 5 mice per group. I-K The disease scores of EAE mice treated with vehicle or BrQ (30 mg/kg) daily by gavage starting from day 7, day 14, or day 21 after immunization. n = 5–6 mice per group **p < 0.01, ****p < 0.0001 by two-way ANOVA. The
experiments were repeated twice with consistent results

were qualified, 1–3 µg total RNA of each sample wasused as the starting material to construct a transcriptome sequencing library according to the instructions
of VAHTS Universal V6 RNA-Seq Library Prepkit for
Illumina (NR 604-01/02). The cDNA sequencing on IlluminaHiseq4000 sequence platform was performed byAnnoroad Gene Technology Co., Ltd (Beijing, China).
The raw data were first under quality control processedwith fastp (0.20.0). Subsequently, hisat2 (version 2.1.0)
and htseq-count (version 0.11.2) were used to map
the quality-controlled samples to the mouse reference
genome and generate the gene count matrix. Differentially expressed genes were determined by DESeq2 package (Bioconductor) and only differentially expressed
genes with fold change > 2 or fold change <-2 that were
statistically significant (p value < 0.05) were selected.

on poly-ornithine plus laminin-coated plates in OPC differentiation medium (DMEM/F12, 1% B27, 1% N2, 10 ng/
ml bFGF, 10 ng/ml PDGF-AA) for 2 days. To induce OPC
differentiation, the cells were cultured in the oligodendrocyte medium (DMEM/F12, 1% B27, 1% N2) with orwithout BrQ (0.31, 0.63, 1.25 or 2.5 µM). After differentiation for 4–6 days, the cells were fixed with 4% PFA for
15 min at room temperature. The cells were subsequently
stained with anti-MBP antibody (Abcam, ab7349, 1:500)
and the Alexa Fluor 647 secondary antibody (Invitrogen,A78947, 1:2000), Hoechst 33342 was used to identify cell
nuclei. For cell imaging, we scanned four pictures perwell and detected the nuclei and MBP-positive cells usinga DMi8 microscope (Leica) and quantitative image analysis was performed by Image J software.

Cuprizone-induced demyelination mouse model

A two-way ANOVA test was used to assess the significance of EAE clinical scores between the vehicle and
BrQ-treated group throughout the disease course. Student’s t-test was applied for the other analyses. All data
are expressed as the mean ± SEM. Only p values < 0.05
were considered statistically significant (*p < 0.05,**p < 0.01, ***p < 0.001, and **** p < 0.0001). Statisticalanalysis was performed using the software GraphPad
Prism 9.0.

C57BL/6 mice (9 weeks) maintained in pathogen-free
conditions were fed with 0.2% (w/w) cuprizone (Sigma)
mixed with ground standard rodent chow. The cuprizone diet was maintained for 5 weeks, thereafter the
cuprizone-infused feed was removed and the mice were
given standard chow. The mice were orally administered
BrQ (30 mg/kg/day) from the fifth week. The treatment
lasted for 1, 2, or 3 weeks. The mice were subsequently
anesthetized with 1.25% tribromoethanol and sacrificed.
The brains were carefully removed, paraffin-embedded,
sectioned, and stained the corpus callosum with LFB for
histopathological analysis. Image-Pro plus software wasused for quantization of the myelin loss in the corpus callosum per section.
OPC differentiation and immunocytochemistry

Neural progenitor cell (NPC)-derived OPCs were
induced by culture of cortical NPCs with OPC differentiation conditions. Briefly, the cerebral cortex was
removed from the E14.5-E17.5 embryonic mouse brain
and mechanically dissociated into single cells by filtering
through 70-µm cell strainers placed on a 50 ml conicaltube, and then the cells were collected by centrifugation
at 500×g for 5 min at 4 °C. The cells were cultured in NPC
medium (DMEM/F12 media, 1% B27, 1% N2, 20 ng/mL
EGF, and 20 ng/mL FGF2 for 3–5 days until neurosphere
formation. The neurospheres were dissociated into single
cells with accutase until passage 3 (P3) NPCs and plated

Statistical analysis

Results
BrQ ameliorates clinical symptoms of EAE mice

To investigate whether BrQ (Fig. 1A) confers protection
against MS, MS animal model EAE was induced in male
C57BL/6 mice by immunization with MOG35–55 peptide
and 3,10 or 30 mg/kg BrQ was given every day via oral
administration from day 3 after immunization, an equalvolume (200 µL) of 0.5% CMC was given as vehicle control. The clinical score for EAE mice was assessed daily
until the end of the experiment (Fig. 1B). The results
showed that BrQ displayed a slight but statistically beneficial effect in alleviating disease severity at low dose of
3 mg/kg body weight. 10 mg/kg BrQ can markedly inhibit
the disease severity of EAE and reduce disease incidencecompared with the vehicle group. Encouragingly, 30 mg/
kg BrQ administered by gavage resulted in a lower tonearly less than 50% morbidity, as well as a dramaticallyreduction in disease severity (Fig. 1C, D). Taken together,
our results showed that BrQ not only markedly lowered

Yang et al. Journal of Neuroinflammation(2024) 21:181

Fig. 2 (See legend on next page.)

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Page 8 of 18(See figure on previous page.)
Fig. 2 BrQ does not display any effect on OPC differentiation and myelin remyelination. A Schematic diagram for the generation of OLs from cortical
NPCs isolated from mouse E15.5 embryos. B Representative immunofluorescent staining images of the OL marker MBP+ cells in OPCs induced to differentiate toward OLs in the presence of various concentrations of BrQ for 4.5 days. The percentage of MBP+ cells was quantified. Scale bars, 100 μm. C
Schematic illustration of the cuprizone-induced demyelination mice model and BrQ treatment. Male adult C57BL/6 mice were induced demyelination by
feeding with a diet that contained 0.2% (w/w) cuprizone for 5 weeks. Following cuprizone withdrawal, the mice were treated with 0.5% CMC solution orBrQ (30 mg/kg, oral administration) for 1, 2, or 3 weeks. D Representative images of the corpus callosum region stained with Luxol fast blue after cuprizone
and BrQ treatment. Scale bars, 200 μm. E Statistical analysis of the myelinating areas in D (n = 5–6 mice per group). Data are shown as mean ± SEM andanalyzed by unpaired two-tailed t-test. The experiments were repeated twice with consistent results

the peak severity and cumulative clinical score of EAE
mice in a dose-dependent manner but also delayed theonset and reduced disease incidence.
Histological analysis of spinal cord was performed atday 18 post-immunization. To assess leukocyte infiltration and myelin loss in the CNS, the lumbar spinal cords
with the highest leukocyte infiltration and the most
severe demyelination were acquired. We found that BrQ
treatment leads to a dramatic reduction of inflammatoryfoci and leukocyte infiltration in the spinal cord than thevehicle group according to H&E staining result (Fig. 1E,
F). Luxol fast blue staining also displayed less extensive
demyelination in the white matter region of the spinal
cord in BrQ-treated EAE mice (Fig. 1G, H).
To further gain insight into the mechanism underlying BrQ-mediated protection against EAE, we treated
EAE mice with BrQ on different days during the diseaseprogression until the end of this study. Our results indicated that treatment with BrQ, started before the onset ofsymptoms, significantly reduced the severity of the disease (Fig. 1I). More interestingly, an intervention protocol in which BrQ treatment was started after the onset ofsymptoms (day 14, when EAE clinical score reached 1),or the remission stage (day 21) also showed remarkablecurative effect (Fig. 1J, K). Taken together, all these data
demonstrate the preventative and therapeutic benefit of
BrQ for EAE.
BrQ does not promote OPC towards OL differentiation andmyelin remyelination in cuprizone-induced demyelination
mouse model

Considering that BrQ treatment had such a striking palliative effect on EAE mice, we wondered whether BrQ
could simultaneously and directly promote myelin regeneration. Oligodendrocyte precursor cells (OPCs) migrate
throughout the brain and spinal cord where they proliferate and differentiate into oligodendrocytes (OLs), OLs
wrap nerve fibers with layers of specialized cell membrane to form myelin sheaths [20, 21]. Thus, we firstly
established an assay to induce OL differentiation fromneural progenitor cells (NPC)-derived OPCs to assess theeffect of BrQ in promoting OPC differentiation towards
OL (Fig. 2A). Mice E15.5 embryos were used to obtaincortical neurospheres, which could be propagated andpassaged in NPC medium. NPCs were then induced into
OPCs in OPC medium supplemented with bFGF and

PDGF-AA. After removal of growth factors, OPCs differentiated spontaneously into OLs in the presence of different concentrations of BrQ for 4.5 days, and then the cells
were fixed and subjected to immunofluorescence staining
for MBP, a marker of mature OLs. The quantification data
showed that the fraction of MBP+ cells did not increase
with BrQ concentration, suggesting BrQ may have noeffect on OL differentiation from OPC or myelin sheathrepair in vitro (Fig. 2B).
To further evaluate the efficacy of BrQ in enhancing
remyelination, we used the widely recognized cuprizone
induced demyelination model [22]. Nine-week-old adultmale C57BL/6 mice were fed 0.2% (w/w) cuprizone dietfor 5 weeks, followed by standard chow and daily oral
administration of BrQ (30 mg/kg) or vehicle for 1–3
weeks (Fig. 2C). The myelin status in the corpus callosum
region was assessed by Luxol fast blue staining, a copper phthalide dyestuff, which has the property of binding
to the myelin sheath in an alcoholic solution. The result
showed that cuprizone treatment resulted in an almost
complete loss of myelin in the corpus callosum comparedwith normal C57BL/6 mice. After cuprizone removal,spontaneous myelin repair could be gradually observedin the vehicle and BrQ groups at 1, 2 and 3 weeks. However, BrQ treatment did not substantially accelerated theremyelination course (Fig. 2D, E). Overall, The findings
indicate that BrQ could not promote myelin regeneration
both in vitro and in vivo.
BrQ promotes activation and proliferation of CD8+ T cells in
EAE mice

To comprehensively explore the underlying mechanismsof BrQ-mediated amelioration of EAE symptoms, we
performed bulk RNA-seq analysis of lymphocytes inthe dLN on day 10 after immunization in the EAE micetreated with 0.5% CMC solution or 30 mg/kg BrQ. As
shown in the volcano diagram (Fig. 3A), we identified
2347 differentially expressed genes compared to the vehicle group (fold change > 2, p < 0.05), among which 1344
genes were upregulated, and 1003 genes were downregulated. Further GO (Gene ontology) enrichment analysis
revealed that the gene categories significantly affectedby BrQ in the dLN lymphocytes cells were associatedwith activation of immune response, activation of innate
immune response, response to interferon-beta, cellularresponse to interferon-beta and positive regulation of

Yang et al. Journal of Neuroinflammation(2024) 21:181

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Fig. 3 BrQ prominently activates and promotes the proliferation of CD8+ T cells in vivo. The leukocytes isolated from the dLN of EAE animals treated with
0.5% CMC solution or 30 mg/kg BrQ on day 10 post-immunization were performed bulk RNA-seq. A Volcano plot represents the differential expressedgene between BrQ and vehicle groups. Genes which were differed significantly compared to the vehicle group were colored with red (highly expressedin BrQ group) or blue (highly expressed in control group). B Gene ontology analysis of BrQ group significant gene. C KEGG analysis of BrQ group significant genes. D-G GSEA maps of the major differential gene sets in the vehicle and BrQ groups. H, I Circle plots and heatmap showing inferred cell-cellcommunications (ligand–receptor pairs) between any two immune cell subsets in dLN. Different colors in the circle plot represent different cell groups
and the thickness of the line represents the strength of communication between the two cells, with thicker lines indicating stronger communication(interactions). In the heatmap, the vertical coordinate is the ligand, which corresponds to the arrow beginning cell type in the circle plots; the horizontal coordinate is the receptor, which corresponds to the arrow pointing to the cell type. The color in each cell represents the relative communication
strength. J KEGG pathway analysis of CD8+ T cells from leukocytes isolated from the dLN. K Representative FACS images and statistics of CD8+ T cells in theleukocytes of dLN from EAE mice treated with BrQ (30 mg/kg) or 0.5% CMC solution on day 10 after immunization. L The expression of IFN-γ in the CD8gate was analyzed by intracellular staining. Data are presented as mean ± SEM. *p < 0.05, ***p < 0.00 versus vehicle group (n = 5–6 per group, Student’s ttest). The experiments were repeated 2–3 times with consistent results

defense response (Fig. 3B). Similarly, T cell receptor signaling, CD28 signaling in T helper cells and Interferonalpha/beta signaling pathway also stood out according
to KEGG (Kyoto Encyclopedia of Genes and Genomes)
pathway analysis (Fig. 3 C). Consistent with the above
GO and KEGG results, the GSEA (Gene Set Enrichment
Analysis) analysis also showed that activation of immune

response, cytokine production, innate immune response
and positive regulation of defense response were upregulated, indicating that BrQ extensively activates immune
cells in the dLN (Fig. 3D-G). We then employed CellChat
analysis to investigate which immune cells are activatedand contribute to alleviating EAE clinical symptoms. The
results showed that activation of CD8+ T cells was mostYang et al. Journal of Neuroinflammation

Fig. 4 (See legend on next page.)(2024) 21:181

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Yang et al. Journal of Neuroinflammation(2024) 21:181

Page 11 of 18(See figure on previous page.)
Fig. 4 BrQ alleviates EAE symptoms in a CD8+ T cell-dependent manner. A Diagram illustrating the administration scheme of anti-CD8 or an isotype
control in EAE mice. The EAE mice were treated daily by gavage with either 200 µL of vehicle (0.5% CMC) or 30 mg/kg of BrQ, starting from day 3 postimmunization. B The EAE clinical scores were assessed in mice treated with vehicle or BrQ (30 mg/kg), with or without CD8 depletion. ####p < 0.0001 by
two-way ANOVA. C Statistical analysis was performed on the EAE onset day, peak EAE score, and cumulative clinical scores (n = 6–8 per group). Data aremean ± SEM. *p < 0.05, **p < 0.01 (one-way ANOVA test). D-E The number of Th1 and Th17 cells infiltrating the CNS were quantified in mice treated withvehicle or BrQ (30 mg/kg), with or without CD8 depletion, n = 6 per group. Data are mean ± SEM. **p < 0.01, ***p < 0.001, (one-way ANOVA test). F-G The
splenocytes and leukocytes harvested from EAE mice on day 10 postimmunization were restimulated with MOG35–55 (20 µg/ml) for 48 h and 72 h invitro, respectively. Evaluation of recall responses of antigen-specific CD4+ T cell was performed using FACS analysis (n = 6). Data are mean ± SEM. **p < 0.01
(Student’s t test). H Isolated CD8+ T cells from the draining lymph nodes of C57BL/6 mice were labeled with CFSE. The proliferation of CD8+ T cells wasassessed via flow cytometry after treatment with 20 ng/ml IL-2, 5 µg/ml CD3/CD28, and 0.25 µM BrQ for 3 days. I The total leukocytes from C57BL/6 mice
were labeled with CFSE. The proliferation of CD8+ T cells was evaluated by flow cytometry after treatment with 20 ng/ml IL-2, 5 µg/ml CD3/CD28 andvarying concentrations of BrQ for 3 days. Data are mean ± SEM. **p < 0.01, ****p < 0.0001 versus vehicle group by one-way ANOVA test. The experiments
were repeated twice with consistent resultspronounced in peripheral dLN lymphocytes, primarilycollected from the axillary, lateral axillary, and superficial inguinal regions, in BrQ-treated EAE mice. Of note,
we discovered that among these immune cells interacting with CD4+ T cells, CD8+ T cells were the most significantly altered signaling sender and receiver (Fig. 3H,I). In addition, QIAGEN Ingenuity Pathway Analysis(IPA) confirmed that CD8+ T cells activation associatedsignaling pathways, including TCR signaling and interferon gamma signaling, were elevated (Fig. 3J). Consistent with the above findings, our results demonstrated
a substantial increase in the percentage of CD8+ T cells
and CD8+ IFN-γ+ T cells in the dLN of EAE mice in vivo,
compared to the vehicle group (Fig. 3K, L). Moreover,BrQ administration significantly increased the levels of
IFN-γ in serum (Fig. 3 M), while markedly reducing IL17a levels, without affecting the levels of IL-6, TNF, andIL-10 (Supplementary Fig. 2). Altogether, these data
demonstrate that BrQ promotes the remarkable activation and proliferation of CD8+ T cells in EAE mice.
The amelioration of EAE clinical symptoms by BrQ relies on
CD8+ T cells

To determine how BrQ affected the inflammatory process in EAE, we analyzed the percentages of CD4+ T
cells, CD8+ T cells, B cells, or macrophages in the spleenand dLN at day 9 postimmunization by flow cytometry.
The results unveiled that the most prominent variations
occurred in the percentages of CD8+ T cell subpopulations (Supplementary Fig. 1). Next, we aimed to explore
the role of CD8+ T cells in BrQ-mediated alleviation of
EAE clinical symptoms. We used a CD8-specific monoclonal antibody (CD8 mAb) to deplete CD8+ T cells after
immunization. Injection on EAE day 3 and day 4 rapidly
depleted CD8+ T cells, maintaining a 71.2% clearance
rate even after 6 days (almost complete clearance of CD8+
T cells would lead to nearly complete mortality in mice
during the progression of EAE, particularly at the peakof the disease). An additional dose was given on EAE day
11. The clearance efficiency of CD8+ T cell in peripheral
blood on EAE day 15 was 58.5% with minimal impact on
CD4+ T cells, as determined by flow cytometry (Fig. 4A

and Supplementary Fig. 3). Our results revealed that
CD8+ T cell depletion led to a slight exacerbation of EAE
clinical symptoms. Importantly, the beneficial effects of
BrQ disappeared after CD8 depletion, indicating that
BrQ alleviates EAE in a CD8+ T cell-dependent manner (Fig. 4B, C). Notably, BrQ also reduces Th1 and Th17
cell infiltration into the CNS in a CD8+ T cell-dependent
manner (Fig. 4D, E). To test whether the active suppression induced by expanded CD8+ T cells was directed
toward antigen-specific CD4+ T cells. We isolated lymphocytes from the dLN and spleen of EAE mice treatedwith 0.5% CMC solution or 30 mg/kg BrQ on day 10
after immunization and examined the recall responses ofthese cells to MOG35 − 55 in vitro. Our results confirmedthat the percentages of MOG35 − 55-reactive CD4+ T cell
subpopulation in the dLN and spleen were dramaticallyreduced compared with the vehicle group (Fig. 4F, G).
To further investigate whether BrQ directly promotes
CD8+ T cell proliferation, we obtained lymphocytes
from dLN, isolated CD8+ T cells by negative selection,and labeled with CFSE. Then, different concentrationsof BrQ were added and CD8+ T cell proliferation wasassessed after culture for 3 days (cell apoptosis began tooccur at 0.5 µM BrQ). The results showed that BrQ did
not directly promote CD8+ T cell proliferation (Fig. 4H).
However, the addition of BrQ to total lymphocytes significantly promoted CD8 expansion, suggesting that
BrQ might induce CD8+ T cell proliferation indirectly
by acting on other immune cells (Fig. 4I). In summary,our findings demonstrate that BrQ promotes CD8+ T
cell proliferation indirectly, leading to the suppression of
antigen-specific CD4+ T cells and ultimately alleviating
EAE in a CD8+ T cell-dependent manner.
BrQ restricts Th1 and Th17 development in vivo

Moreover, Th1 and Th17 cells are long considered to be
the major effectors and culprits in a plethora of autoimmune and inflammatory diseases including multiple sclerosis [23–25]. Thus, we turned our attention to explore
whether BrQ alleviated the disease severity of EAE micethrough activated CD8+ T cells mediated Th1 and Th17
inhibition in vivo. Firstly, we analyzed the number ofYang et al. Journal of Neuroinflammation(2024) 21:181

inflammatory cells infiltrating into the CNS at day 18
post-immunization. The results demonstrated that BrQ
led to a significant decrease in total leukocyte infiltrationin the CNS compared with the vehicle group (Fig. 5A).

Page 12 of 18

We further assessed infiltrating CD4+ cell in the spinal
cord and brain by FACS analysis. Consistent with theabove observation on total leukocyte infiltration in EAE
mice, BrQ treatment also caused a remarkable reduction

Fig. 5 BrQ blocks Th1 and Th17 cells in EAE mice. A Total CNS infiltrate lymphocytes were isolated from EAE mice treated with 0.5% CMC solution or BrQ(30 mg/kg, oral, starting from day 3) on day 18 post-immunization and quantified by flow cytometry. B Flow cytometric analysis of CD4+ T cells in the
CNS infiltrate lymphocytes. C Statistical data of absolute numbers of infiltrating CD4+ T cells within the CNS. D Absolute numbers of Th1 and Th17 cells in
the infiltrated CD4+ T gate. E, G Leukocytes were isolated from the draining lymph node (dLN) of EAE animals treated with 0.5% CMC solution or 30 mg/
kg BrQ on day 10 post-immunization. Th1 and Th17 cells were analyzed by intracellular staining of IFN-γ and IL-17 A in the CD4 gate, respectively. F, H
Statistical analysis of absolute numbers of Th1 and Th17 cells in dLN (n = 5–6 per group). Data represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001versus vehicle group (Student’s t test). The data are a representative for two independent experiments

Yang et al. Journal of Neuroinflammation(2024) 21:181

in CD4+ T cell number and percentage (Fig. 5B, C). In
addition, the number of CD4+IFN-γ+ and CD4+IL17A+
T cells in the BrQ treated group were less than in vehicle treated mice (Fig. 5D). However, although BrQ promoted the proportion of Treg cells infiltrating the CNS,
the absolute number of Treg cells did not increase due tothe reduced total number of CNS-infiltrating cells (Supplementary Fig. 5C, D). These findings indicated that BrQ
treatment reduced the infiltration of inflammatory cells
into the central nervous system, especially the number ofTh1 and Th17 cells.
Furthermore, we analyzed the profile of MOG-reactive
CD4+ T cells in the peripheral draining lymph node of
BrQ-treated and untreated mice at day 10 post-immunization. The results revealed that the proportion of Th1
and Th17 cells in the CD4+ population were dramatically reduced (Fig. 5E, G). Consistent with the observation in the CNS, BrQ treatment also caused a remarkabledecrease in the total number of Th1 and Th17 cells in
the lymphocytes of the draining lymph node (Fig. 5F, H).
Moreover, BrQ significantly increased the proportion andtotal number of Treg cells in the dLN (Supplementary
Fig. 5A, B). Nevertheless, FACS analysis revealed thatBrQ did not influence the differentiation of Th1, Th17,
or Treg in vitro (Supplementary Fig. 4). Taken together,
these data drive us to conclude that BrQ reduces Th1 andTh17 frequencies and EAE immunopathology by promoting CD8+ T cell activation and proliferation.
No immunotoxicity signs were observed in BrQ-treated
C57BL/6 mice

To assess the safety of BrQ administration in vivo, we
assessed the safety of BrQ in vivo. Normal male C57BL/6
mice (7–8 wk) were gavaged with 200 µL 0.5% CMC solution or different dosages of BrQ (3, 10 or 30 mg/kg) dailyfor 3 weeks and their body weight was weighed weekly.
The data showed there were no significant alterations
in C57BL/6 mice treated with various dosages of BrQ(Fig. 6A). Moreover, there were no discernible changes in
spleen size, morphology, or weight (Fig. 6B, C), indicatingthat BrQ administration at the macroscopic level had no
significant effect on normal growth and spleen health inmice.
Considering that the lymphatic and circulatory systems are critical in maintaining immunological stability and safeguarding the body from disease [26]. We
next validated whether the administration of BrQ had
any toxic effects on the major immune cell populationsin the draining lymph nodes and bloodstream. Leukocytes from the draining lymph node and blood of mice
were collected after the BrQ administration continued
for 21 days and subjected to flow analysis for CD4, CD8,
B220, and CD11b cells to differentiate among T cells, B
cells, and macrophages/neutrophils. The results showed

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that the percentage of these cells remained unaltered(Fig. 6D-G). It has been recognized that T cells, particularly CD4+ T cells, play a crucial role in MS [27]. Furthermore, given that the thymus provides a nurturing site forthe differentiation, development, and selection of T cells[28, 29], the effect of BrQ on T cell development in thethymus was also evaluated. As a result, the percentage of
CD4+, CD8+, CD+CD8+ (DP) and CD4−CD8− (DN) in
the thymus also remained unchanged (Fig. 6H, I). Theseobservations lead us to conclude that BrQ did not exert
significant immunotoxicity and provided strong support
for its safety in further research and potential clinicalapplications. Altogether, these data suggested that BrQ
administration induces negligible immunotoxicity, supporting its safety in further research and potential clinicalapplications.

Discussion
As the most prevalent non-traumatic neurological disease among young adults, MS has been a rapidly growing global health concern [30]. Despite the introductionof various disease-modifying therapies in recent years, acomplete clinical cure for MS remains elusive, with currently available treatments only capable of slowing disease progression. Consequently, it is urgently needed
to develop safe and efficacious drugs targeting multiple
sclerosis [31]. In our study, we observed that mice treatedwith 2-bromo-1,4-naphthalenedione (BrQ), a derivativeof naphthalenedione, exhibited reduced severity of EAE
symptoms, including lower clinical scores, decreased leukocyte infiltration, and less extensive demyelination in
the CNS. Through comprehensive analysis of cell interactions using bulk RNA-seq data and validation throughflow analysis, we discovered that BrQ significantly
enhances the expansion of CD8+ T cells and their interactions with other immune cells in peripheral immune
tissue in EAE mice. Further experiments involving CD8+
T cell depletion confirmed that BrQ alleviates EAE in a
CD8+ T cell-dependent manner. Mechanistically, we
found that expanded CD8+ T cells selectively reduce
antigen-specific CD4+ cells, subsequently inhibiting thedevelopment of Th1 and Th17 cells in vivo, ultimately
leading to relief from EAE. Our findings shed light on theunique therapeutic potential of BrQ for the treatment ofMS (Fig. 7).
Substantial evidence suggests that CD8+ T cells are theprimary immune cell population within CNS lesions ofMS patients, indicating their pivotal role in the diseaseprogression of MS [27, 32]. Both in MS patients and the
EAE animal model, clonally expanded CD8+ T cell populations effectively reduce disease severity by actively suppressing autoreactive CD4+ T cells. Conversely, depletionof CD8+ T cells exacerbates EAE symptoms [33–35].
Moreover, nonclassical MHC class Ib–restricted CD8+

Yang et al. Journal of Neuroinflammation(2024) 21:181

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Fig. 6 No signs of immunotoxicity are observed in BrQ-treated mice. Normal 8-week-old C57BL/6 mice were administered orally with 200µL vehicle (0.5%
CMC) or BrQ (3, 10, or 30 mg/kg/day) for 21 days. A Body weight was monitored every week. B, C Spleen samples were collected, pictured and weighed.
D, E Leukocytes in the draining lymph node were isolated and stained for CD4+ T cells, CD8 + T cells, B cells (B220) and CD11b+ cells. Cell percentages were
determined with flow cytometry analysis. F, G Cell surface staining results and statistic data of the CD4+ T cells, CD8+ T cells, B cells (B220) and CD11b+
cells in the blood. H, I Flow cytometric analysis and quantification of CD4+, CD8+, CD4−CD8− (double negative, DN) and CD4+CD8+ (double positive, DP)
in the thymus. Data are mean ± SEM of 5 mice per group versus vehicle group (one-way ANOVA). Representative data of two independent experiments

Yang et al. Journal of Neuroinflammation(2024) 21:181

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Fig. 7 Schematic diagram of the protective mechanism of BrQ against EAE pathogenesis

T cell (HLA-E in humans and Qa-1b in mice), CNS-specific CD8+ T, CD8+CD25+Foxp3+, CD8+CD44+Ly49+,
CD8+CD28−, CD8+CD122+ and KIR+CD8+ T cells havebeen suggested to have a regulatory function [36–39].
In contrast, IL-17 A secreting CD8+ T cells and myelin
antigen-specific CD8+ T cells were demonstrated to support EAE symptoms [40, 41]. Given the heterogeneityand complexity of CD8+ T cell subpopulations, none of
the twenty-three disease-modifying therapies approvedby the U.S. Food and Drug Administration was specifically developed to target CD8+ T cells for treating MS.
Although glatiramer acetate mitigates EAE to someextent by inducing Qa-1-restricted regulatory CD8+ T
cells. Notably, CD8+ T cells were identified as primary
signaling mediators in the peripheral immune system of
BrQ treated EAE mice. Importantly, we observed that
BrQ activates and amplifies CD8+ T cells within the
spleen, dLN and blood, leading to a significant reductionin the MOG35 − 55-reactive CD4+ T cell subset in EAE
mice. CD8+ T cell depletion confirmed the efficacy of
BrQ was exerted in a CD8+ T cell-dependent manner.
Furthermore, we observed the activation of 4-1BB signaling in CD8+ T cell after BrQ treatment, while theagonistic anti-4-1BB monoclonal antibody can suppress
antigen-specific CD4+ T cells by activating CD8+ T cells
in rheumatoid arthritis animal models [42]. We inferredthat BrQ largely promotes the proliferation of CD8+ T
cells by activating 4-1BB. Likewise, despite reduced CD8+
T cell levels in the blood on day 15 in EAE mice, asymptomatic BrQ-treated mice remained symptom-free evenafter BrQ administration cessation for 1 month (unpublished observation). We speculated that expanded CD8+
T cell not only exerts immunosuppressive effects but also
potentially develop into antigen-specific memory T cell.
Thus, BrQ may represent the first small molecule candidate drug capable of specific increasing CD8+ T cells with

Yang et al. Journal of Neuroinflammation(2024) 21:181

suppressive function, thereby providing novel therapeutic strategies for EAE and other autoimmune diseases.
It has been well established that activation of peripheral autoreactive effector CD4+ T cells mediate disease progression and severity [42]. Moreover, both
IL-17 A-producing Th17 cells and IFN-γ-producing
Th1 cells are recognized as pivotal players in driving thepathogenesis of EAE [43]. Upon activation, CD8+ T cells
produce substantial amounts of IFN-γ, allowing themto modulate autoimmune responses by suppressing Th1
and Th17 cells in an IFN-γ-dependent manner [44]. Our
results showed that BrQ boosts IFN-γ production in
CD8+ T cells in vivo, along with increased serum levelsof IFN-γ. As reported, IFN-γ deficiency exacerbates EAE[45]. The function of IFN-γ in regulating Th1 and Th17
responses have been well elucidated. First, IFN-γ limits
Th1 responses by converting IFN-γ-producing Th1 cells
into IL-10-producing type 1 regulatory T (Tr1) cells [46].
Secondly, IFN-γ directly inhibits naive T cells from differentiating into Th17 cells. Instead, the inhibition of IFN-γ
signaling enhances pathogenic Th17 effector cell development, worsening autoimmunity [47]. IFN-γ also indirectly inhibits the pathogenic Th17 response by inducing
dendritic cells to secrete IL-27 [48]. Thirdly, coculture
with activated CD8+ T cells markedly enhances apoptosis of Th1 and Th17 cells in vitro. Additionally, activatedCD8+ T cells can directly eliminate effector/autoreactive
CD4+ cells by releasing cytolytic granules like granzyme
B and perforin, leading to direct lysis [37, 44]. Furthermore, our research supports these findings by demonstrating that BrQ restrains Th1/Th17 cell development inboth peripheral dLN and CNS in vivo. Collectively, ourstudy suggests BrQ mediates the activation and expansion of CD8+ T cells to suppress Th1/Th17 development,
thereby conferring protection against EAE/MS.
MS are characterized by autoimmune-mediated demyelination and neurodegeneration. The treatment of multiple sclerosis currently has two main goals: controlling the
inflammatory activity and promoting remyelination. Of
these, remyelination is a promising strategy for treating
MS as it restores damaged CNS regions [49–51]. Accordingly, we next verified whether BrQ could directly promote myelin regeneration in vivo and in vitro. Our study
indicated that BrQ does not stimulate OPC differentiation into mature oligodendrocytes that remyelinate axons
and myelin sheaths for remyelination in vitro, nor doesit have a direct effect on remyelination in a cuprizoneinduced demyelination mouse model. We speculated
that BrQ appears to modulate EAE pathology throughan immunosuppressive effect rather than by directly
promoting myelin regeneration. However, the inflammatory state of the peripheral immune system contributes to inefficient remyelination in MS [52]. Hence, BrQ,
with its unique mechanism and notable efficacy, offers

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a promising approach when combined with agents promoting remyelination to achieve the goal of curing MS.
However, our study has several limitations. Firstly, BrQ
does not directly promote CD8+ T cell proliferation,
but its effects on immune metabolic stability, cytokine
production, and functionality need further investigation. Secondly, it’s unclear whether BrQ-activated CD8+
T cells require IFN-γ to inhibit Th1 and Th17 frequencies and the exact mechanisms by which CD8+ T cells
limit the encephalitogenic response in BrQ-treated
EAE. Thirdly, although our findings indicate that BrQ
promotes the proliferation of CD8+ T cells indirectlyin vitro. However, it remains uncertain whether BrQ is
metabolized into other substances in vivo or undergoes
metabolic transformation by intestinal microorganisms
into different products, directly promoting the proliferation of CD8+ T cells or alternatively exerting a protective
effect on EAE by modulating intestinal immune cells.
Lastly, the timing of BrQ’s effectiveness on EAE varies
at different disease stages. Aside from BrQ’s potentiallyunique mechanism of action, we speculate that differences in underlying pathology, particularly the differential response rates of CD8+ T cells at various stages of thedisease, result in varying responsiveness to BrQ at different stages. This will be the focus of our forthcoming
research.
In summary, our study demonstrates that BrQ could bea unique molecule drug to amplify specially CD8+ T cellswith suppressive function. These expanded CD8+ T cells
specifically suppress pathogenic CD4+ T cells and Th1/
Th17 development, ultimately resulting in relief from
EAE. Thus, BrQ might present a promising treatment for
MS and other autoimmune diseases in the future.
Abbreviations
BBB	Blood-brain barrier
BrQ	2-bromo-1,4-naphthalenedione
CNS	Central nervous systemdLN	draining lymph nodes
EAE	Experimental autoimmune encephalomyelitis
H&E	Hematoxylin and eosin
LFB	Luxol Fast Blue
MOG	Myelin oligodendrocyte glycoprotein
MS	Multiple sclerosis
OPC	Oligodendrocyte precursor cells
TCMs	Traditional chinese medicinesTh1	T helper 1 cells
Th17	T helper 17 cells
Treg	regulatory T cells

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03172-x.
Supplementary Material 1

Yang et al. Journal of Neuroinflammation(2024) 21:181

Page 17 of 18

Acknowledgements
Home for Researchers editorial team (www.home-for-researchers.com) for
language editing service. Graphical design was performed using biorender
online software (https://www.biorender.com/).
Figure 7 depicts an anatomical diagram of draining lymph nodes used in ourstudy sourced from the literature [53]8.

Author contributions
H.Q., X.R. and W.J. supervised this project. C.X. design the experiments withthe support from AP.X and F.W. C.X. and Q.Y. performed the experiments. Y.C.
analyzed the bulk-seq data. C.X. wrote the initial draft of the manuscript. Y.L.,
Y.T., S.M., C.J., J.S., C.J. carried out some experiments and helped with data
analysis. All authors read and approved the final manuscript.

10.

Funding
This study was supported by grants from the National Natural Science
Foundation of China (82101879, 82272605, 81972151) and China Postdoctoral
Science Foundation (2020M671146).

13.

Data availability
No datasets were generated or analysed during the current study.

Declarations

9.

11.
12.

14.
15.
16.

Ethics approval and consent to participate
All animal experiments were carried out according to guidelines approved by
the Institutional Animal Care and Use Committee of Sun Yat-sen University. No
human study was performed.

17.

Consent for publication
Not applicable.

19.

Competing interests
The authors declare no competing interests.

20.

Author details
1
Central Laboratory, Chaozhou Central Hospital Affiliated to Southern
Medical University, Chaozhou, Guangdong Province, China
2
Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun
Yat-sen University, Guangzhou, China
3
Department of Rehabilitation Medicine, The Third Affiliated Hospital, Sun
Yat-Sen University, No. 600 Tianhe Road, Guangzhou, China
4
Key Laboratory for Stem Cells and Tissue Engineering, Sun Yat-sen
University, Ministry of Education, Guangzhou, China
5Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for
Stem Cells and Tissue Engineering, Sun Yat-Sen University, Ministry of
Education, Guangzhou, China
6
Department of Biochemistry, Zhongshan School of Medicine, Sun YatSen University, Guangzhou, China
7
Shanghai Institute of Immunology, Shanghai Jiao Tong University Schoolof Medicine, Shanghai, China
Received: 24 April 2024 / Accepted: 11 July 2024

18.

21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.

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