THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 34, pp.

28656 –28665, August 17, 2012

Author’s Choice © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

The Antiepileptic Drug Valproic Acid Restores T Cell

Homeostasis and Ameliorates Pathogenesis of Experimental
Autoimmune Encephalomyelitis*
Received for publication, February 26, 2012, and in revised form, June 20, 2012 Published, JBC Papers in Press, June 25, 2012, DOI 10.1074/jbc.M112.356584
Jie Lv‡§1, Changsheng Du‡1, Wei Wei‡§, Zhiying Wu¶, Guixian Zhao¶, Zhenxin Li¶, and Xin Xie‡§2
From the ‡Shanghai Key Laboratory of Signaling and Disease Research, Laboratory of Receptor-based Bio-medicine, School of Life
Sciences and Technology, Tongji University, Shanghai 200092, China, the §State Key Laboratory of Drug Research, the National
Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China, and the
¶
Department of Neurology and Institute of Neurology, Huashan Hospital, Shanghai Medical College, Fudan University,
Shanghai 200040, China
Background: Dysregulation of T cell survival and apoptosis is the common cause of autoimmune diseases including
multiple sclerosis (MS).
Results: Valproic acid (VPA) treatment restores the dysregulated apoptosis of T cells and reduces the symptoms of EAE.
Conclusion: In addition to the antiepileptic activity, VPA also regulates T cell homeostasis.
Significance: As an orally available drug, VPA might be used to treat autoimmune diseases, such as MS.

Maintaining a constant number and ratio of immune cells is tightly regulated to ensure the effective elimination of the anti-
one critical aspect of the tight regulation of immune homeosta- gen in question but no severe tissue damage or death of the
sis. Breakdown of this balance will lead to autoimmune diseases organism. Maintaining a constant number and ratio of immune
such as multiple sclerosis (MS). The antiepileptic drug valproic cells is one aspect of such tight regulation (2).
acid (VPA) was reported to regulate the growth, survival, and Multiple sclerosis (MS)3 is an autoimmune disease charac-
differentiation of many cells. However, its function in T cell terized by the immune-mediated demyelination and neurode-
homeostasis and MS treatment remains unknown. In this study, generation of the CNS (3). Experimental autoimmune enceph-
VPA was found to reduce spinal cord inflammation, demyelina- alomyelitis (EAE) is an animal model that shares many
tion, and disease scores in experimental autoimmune encepha- pathological and histological similarities with MS. Although
lomyelitis, a mouse model of MS. Further study indicated that many mechanisms have been proposed for the pathogenesis of
VPA induces apoptosis in activated T cells and maintains the MS, including oxidative stress, excitotoxicity, autoimmunity,
immune homeostasis. This effect was found to be mainly medi- hormonal imbalance (4), etc., it is generally accepted that the
ated by the caspase-8/caspase-3 pathway. Interestingly, this overactivation of CD4⫹ T cells, especially the TH-1 and TH-17
phenomenon was also confirmed in T cells from normal human subpopulations, is the direct cause of this disease (5). Recently,
subjects and MS patients. Considering the long history of clini- CD8⫹ T cells have also been implicated in the pathogenesis of
cal use and our new findings, we believe VPA might be a safe and MS (6, 7). Previous studies suggest two major ways to maintain
effective therapy for autoimmune diseases, such as multiple immune homeostasis. The first way involves the inhibitory
sclerosis. cytokines, which limit the strength of immune cell activation
and expansion, such as TGF-␤ or IL-10 (8, 9). Up-regulation of
IL-10 production by small molecules or repetitive antigen stim-
The immune system protects the body from disease-causing ulation of effector TH-1 cells in the presence of TGF-␤ has been
microorganisms. Foreign antigen stimulation usually leads to reported to suppress EAE (10, 11). The second way is to control
specific immune responses characterized by immune cell acti- cell death. Molecules such as Fas, Bim, Bax, and the caspases-8
vation, differentiation, proliferation, and inflammatory gene and -10 are critically involved (12, 13). A recent study reported
expression, all of which in turn lead to inflammation. After the that enhanced expression of Bcl2 via ␤-arrestin 1-dependent
removal of antigens, the immune system returns to its preacti- epigenetic modification attenuated the death of activated
vation state, ready to respond to next wave of attacks (1). The CD4⫹ T cells and thus led to a severe form of EAE (14).
strength and the duration of the immune responses must be Histone deacetylase inhibitors (HDACis) are compounds
that interfere with the function of histone deacetylase, an
important class of epigenetic modulators. HDACis such as val-
* This work was supported by National Natural Science Foundation of China proic acid (VPA) have a long history of use in psychiatry and
Grants 31000399, 31171348, 31071227, and 81100963, 973 Program Grant
2012CB910404), and the Ministry of Science and Technology of China
3
Grant 2009ZX09302-001. The abbreviations used are: MS, multiple sclerosis; VPA, valproic acid; EAE,
Author’s Choice—Final version full access. experimental autoimmune encephalomyelitis; HDACi, histone deacety-
1
These authors contributed equally to this work. lase inhibitor; CFSE, carboxyfluorescein diacetate succinimidyl ester;
2
To whom correspondence should be addressed: 189 Guo Shou Jing Rd., Z-VAD-FMK, Z-Val-Ala-Asp (OMe)-fluoromethyl ketone; PBMC, peripheral
Shanghai 201203, China. Tel.: 86-21-50801313, Ext. 156; Fax: 86-21- blood mononuclear cell(s); PI, propidium iodide; ANOVA, analysis of
50800721; E-mail: xxie@mail.shcnc.ac.cn. variance.

28656 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 34 • AUGUST 17, 2012
 VPA Ameliorates EAE Pathogenesis
neurology as mood stabilizers and anti-epileptics (15, 16). 2, paresis (weakness, incomplete paralysis of one or two
Recently, two HDACis, vorinostat and romidepsin, were hindlimbs); 3, paraplegia (complete paralysis of both
approved to treat cutaneous T cell lymphoma (17, 18). HDACis hindlimbs); 4, paraplegia with forelimb weakness or paralysis;
can induce high rates of apoptosis in many cell lines of hema- and 5, moribund state or death. For drug treatment, VPA was
tological malignancy. The death receptor pathway and mito- given via intraperitoneal injection (100 –300 mg/kg of body
chondria/caspase pathway are both believed to be involved in weight) or oral administration (10 –300 mg/kg of body weight)
HDACi-induced apoptosis (19). Such proapoptotic activity also once daily from day 3 or 12 postimmunization until the end of
leads to the investigation of HDACis in the inflammatory dis- the study. Water was given as vehicle control (100 ␮l/mouse).
eases (20). In this study, we examined the effects of VPA on EAE Histopathological and Immunofluorescent Analysis—The
neuropathology. Oral or intraperitoneal administration of VPA mice were anesthetized and perfused with PBS (pH 7.4) fol-
reduced spinal cord inflammation, demyelination, and overall lowed by 4% (w/v) paraformaldehyde. Spinal cord samples were
clinical symptoms of EAE. Mechanism study indicated that then fixed in 4% (w/v) paraformaldehyde overnight. Paraffin-
VPA induces apoptosis in activated T cells and maintains the embedded sections of spinal cord were stained with hematox-
immune homeostasis. This effect was found to be mainly medi- ylin and eosin or with Luxol fast blue for analysis of inflamma-
ated by the caspase-8/caspase-3 pathway. This phenomenon tion or demyelination, respectively. Frozen sections of spinal
was also confirmed in T cells isolated from normal human sub- cord were stained with anti-mouse CD45 or CD4 antibodies
jects and MS patients. Our data suggest that VPA is a safe and and then with appropriate fluorescent-labeled secondary
effective therapy for MS. antibodies.
MATERIALS AND METHODS Isolation and Analysis of CNS Leukocytes Infiltration—Spinal
cords collected after PBS perfusion were homogenized in ice-
Human Samples—Human blood samples were obtained cold tissue grinders and filtered through a 70-␮m cell strainer,
from patients of the outpatient clinic of Huashan Hospital and the cells were collected by centrifugation at 500 ⫻ g for 10
(Shanghai, China) with clinically defined relapsing remitting min at 4 °C. The cells were resuspended in 8 ml of 37% Percoll
MS and healthy volunteers from Tongji University. Informed
and centrifuged onto 4 ml of 70% Percoll cushion in 15-ml tubes
consent was provided, and the sampling was completed in
at 780 ⫻ g for 25 min at 25 °C. The cells at the 37–70% Percoll
accordance with the guidelines of local institutional review
interface were collected and were subjected to flow cytometry
boards.
analysis.
Animals—Female C57BL/6 mice were purchased from
T Cell Proliferation—Naive mouse splenocytes were labeled
Shanghai Laboratory Animal Center (Shanghai, China). All the
with 10 mM carboxyfluorescein diacetate succinimidyl ester
mice were housed in the Tongji University animal care facility
(CFSE) and then cultured for 48 h in complete RPMI 1640
and were maintained in pathogen-free conditions. The mice
medium containing 2 ␮g/ml anti-mouse CD3 antibody, 2
were 8 –9 weeks of age at the initiation of the experiment and
␮g/ml anti-mouse CD28 antibody, and various concentrations
were maintained on standard laboratory chow and water ad
VPA in 96-well plates. The CFSE fluorescence was determined
libitum. All of the experiments were approved and conducted
in accordance with the guidelines of the Animal Care Commit- by flow cytometry.
tee of Tongji University. T Cell Apoptosis—Naive mouse splenocytes were cultured
Reagents—MOG35–55 (MEVGWYRSPFSRVVHLYRNGK) for 48 h in complete RPMI 1640 medium containing 10 ng/ml
with a purity of ⬎95% was purchased from GL Biochem IL-2, 2 ␮g/ml anti-mouse CD3 antibody, and 2 ␮g/ml anti-
(Shanghai, China). Complete Freund’s adjuvant was purchased mouse CD28 antibody in 48-well plates at a density of 3 ⫻
from Sigma-Aldrich. PercollTM and Ficoll-PaqueTM Plus were 106/well. Live cells were purified through the Ficoll (␳ ⫽ 1.077)
purchased from GE Healthcare. FITC anti-mouse CD45, FITC density gradient and restimulated with anti-mouse CD3 (2
anti-mouse CD8a, phycoerythrin anti-mouse CD45R (B220), ␮g/ml) for 24, 48, or 72 h in the presence of various concentra-
phycoerythrin Cy7 anti-mouse CD4, phycoerythrin anti-mouse tions of VPA or the combination of VPA and the caspase inhib-
IL17a, APC anti-mouse IFN-␥, and a BD Cytofix/CytopermTM itor Z-VAD-FMK. Splenocytes isolated from naive or EAE mice
kit were purchased from BD Biosciences. The Dynal威 mouse at day 10 postimmunization were restimulated with MOG35–55
CD4 cell negative isolation kit and Vybrant威 apoptosis assay kit (20 ␮g/ml) for 24, 48, or 72 h in the presence of VPA (1 mM) or
were from Invitrogen. APC anti-mouse CD4/CD8 and APC the combination of VPA (1 mM) and Z-VAD-FMK (10 ␮M).
anti-human CD4/CD8 were purchased from Biolegend (San Human PBMC were isolated from whole blood samples by den-
Diego, CA). sity gradient centrifugation on Ficoll-Paque. PBMC of healthy
EAE Induction and Drug Treatment—Female C57BL/6 mice subjects were cultured in complete RPMI 1640 medium and
8 –9 weeks of age were immunized subcutaneously with 200 ␮g restimulated with 2 ␮g/ml anti-human CD3 and 2 ␮g/ml anti-
of MOG35–55 in complete Freund’s adjuvant containing heat- human CD28 antibodies in 48-well plates for 48 h. Live cells
killed Mycobacterium tuberculosis (H37Ra strain; 5 mg/ml; BD were purified through the Ficoll (␳ ⫽ 1.077) density gradient
Diagnostics). Pertussis toxin (200 ng/mouse; Calbiochem) in and restimulated with anti-human CD3 (2 ␮g/ml) for 24, 48,
PBS was administered intraperitoneally on days 0 and 2. The and 72 h in the presence of various concentrations of VPA or
mice were examined daily for disease signs by researchers the combination of VPA and Z-VAD-FMK. PBMC of MS
blinded to experimental conditions and were assigned scores patients were cultured in complete RPMI 1640 and restimu-
on a scale of 0 –5 as follows: 0, no clinical signs; 1, paralyzed tail; lated with MOG (20 ␮g/ml) for 24, 48, and 72 h in the presence

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VPA Ameliorates EAE Pathogenesis
of VPA (1 mM) or the combination of VPA (1 mM) and Z-VAD- Western Blot—Splenocytes from EAE mice were washed
FMK (20 ␮M). once with PBS and lysed in lysis buffer by sonication for 30 s on
The cells were then stained with propidium iodide (PI) and ice. Protein concentration was determined by the BCA method
annexin V in combination with anti-CD4 or anti-CD8 anti- (Thermo Scientific). The samples were loaded into 12% Tris/
body. The percentage of apoptotic cells (annexin V positive and Gly gels, subjected to SDS-PAGE, and transferred onto a PVDF
PI negative cells) was determined by flow cytometry. membrane (Millipore). Western blotting was performed using
CD4⫹ T Cell Separation and in Vitro Differentiation—Naive the rabbit anti-caspase-3 antibody and corresponding HRP-
CD4⫹ T cells were prepared by magnetic cell separation (Invit- conjugated secondary antibody (Promega) and developed using
rogen) from spleens of female C57BL/6 mice 8 –9 weeks of age. Western Lightning Ultra extreme sensitivity kit (PerkinElmer
The cells were activated with anti-mouse CD3 antibody (2 Life Sciences).
␮g/ml) and anti-mouse CD28 antibody (2 ␮g/ml) and induced Statistical Analysis—The results were expressed as the
to differentiate into TH-1 cells by supplementation with IL-12 means ⫾ S.E. The statistical significance of the EAE clinical
(10 ng/ml) and anti-IL-4 (10 ␮g/ml). For TH-17 differentiation, scores between treatments were analyzed with two-way
cells received anti-IL-4 (10 ␮g/ml), anti-IFN-␥ (10 ␮g/ml), plus ANOVA test. The EAE scores at a given date were analyzed
a TH-17 mixture containing TGF-␤1 (3 ng/ml), IL-6 (30 ng/ml), with a Mann-Whitney U test. Other analyses, including gene
TNF-␣ (10 ng/ml), and IL-1␤ (10 ng/ml). VPA at various con- expression, cell percentage, and histological analysis, were
centrations were added with the cytokine mixture, and the per- assessed by Student’s t test. p ⬍ 0.05 was considered statistically
centages of TH-1 (IFN-␥⫹) and TH-17 (IL-17a⫹) cells were ana- significant.
lyzed with flow cytometry after 72 h of incubation.
To assess VPA-induced apoptosis in TH-1 and TH-17 cells, RESULTS
naive CD4⫹ T cells were induced to differentiate into TH-1 or VPA Ameliorates Clinical Symptoms of EAE—To assess
TH-17 for 72 h. Then the cells were cultured with fresh medium whether VPA has therapeutic effect in MS, EAE was induced in
containing the cytokine mixture in the presence of VPA (1 mM) C57BL/6 mice by immunization with MOG35–55 peptide, and
for another 48 h. The cells were then stained with annexin V in VPA was given once daily via intraperitoneal injection (Fig. 1A)
combination with anti-IFN-␥ or anti-IL-17a antibody. The per- or oral administration (Fig. 1B) from day 3 postimmunization
centage of apoptotic cells (annexin V positive cells) was deter- until the end of the experiment. Water was given as vehicle
mined by flow cytometry. control. VPA displayed dose-dependent inhibition of EAE
Flow Cytometry—Splenocytes, PBMC, or CNS infiltrates severity in both series of experiments. Via intraperitoneal injec-
were incubated for 5 h at 37 °C with phorbol 12-myristate tion, VPA showed a better effect than oral administration at the
13-acetate (50 ng/ml; Sigma), ionomycin (750 ng/ml; Sigma), same dosage; it not only significantly reduced the peak severity
and brefeldin A (10 ␮g/ml; Sigma). Surface markers were and cumulative clinical score of EAE, but also postponed the
stained with relevant antibodies. After surface staining, the cells onset of the disease (Fig. 1, A and B). This is probably due to the
were resuspended in fixation/permeabilization solution (Cyto- higher initial blood concentration of VPA after intraperitoneal
fix/Cytoperm kit; BD Pharmingen), and intracellular cytokine injection than the oral administration. Two low doses of VPA
staining was done according to the manufacturer’s protocol. were also tested via oral administration. At 10 mg/kg, VPA had
Guava easyCyteTM 8HT system and GuavaSoft software were no effect in EAE mice (data not shown), but at 30 mg/kg, VPA
used for the analysis. displayed a slight but statistically significant beneficial effect in
Real Time PCR—Total RNA were extracted from spleno- EAE mice (Fig. 1B). More interestingly, when given after the
cytes using TRIzol reagent (Invitrogen) according to the man- onset of the disease (day 12 postimmunization; Fig. 1C), VPA
ufacturer’s instructions. The RNA was subjected to reverse (300 mg/kg, orally) was still able to reduce the severity of EAE,
transcription with random hexamer primer and Moloney which indicated the therapeutic benefit of this drug in addition
murine leukemia virus reverse transcriptase (Promega). Real to the preventative effect.
time PCR was conducted in the LightCycler quantitative PCR VPA Reduces CNS Leukocyte Infiltration and Neuropathy in
apparatus (Stratagene) using the SYBR Green JumpStartTM EAE Mice—Histological examination of spinal cord was per-
Taq ReadyMixTM kit (Sigma). Expression values were normal- formed at day 17 postimmunization. Compared with vehicle
ized to ␤-actin. The primer pairs used are as follows: caspase-1 treatment, VPA caused a dramatic decrease of leukocyte infil-
sense, 5⬘-CCCCAGGCAAGCCAAAT-3⬘, antisense, 5⬘-GTG- tration in the spinal cord (Fig. 1, D and F). Luxol fast blue stain-
CCATCTTCTTTGTTCTGTTC-3⬘; caspase-3 sense, 5⬘-CTG- ing also revealed less extensive demyelination in VPA-treated
ACTGGAAAGCCGAAACTC-3⬘, antisense, 5⬘-TGGATGAA- EAE mice compared with the vehicle control group (Fig. 1, E
CCACGACCCG-3⬘; caspase-6 sense, 5⬘-AGTACAAGATGG- and G). The CNS leukocyte infiltration was further analyzed by
ACCACAAGAGG-3⬘, antisense, 5⬘-GTTCTTCTGCTCTGA- in situ immunofluorescent staining of the spinal cord sections.
GGTCGTTA-3⬘; caspase-8 sense, 5⬘-GGAAGACATAACCC- Consistent with the results of hematoxylin and eosin staining,
AACTCCG-3⬘, antisense, 5⬘-CTTGTCACCGTGGGATAG- VPA treatment reduced the number of CD45⫹ and CD4⫹ T
GAT-3⬘; caspase-9 sense, 5⬘-GATCAGGGGACATGCAGAT- cells (Fig. 2A) in the spinal cord sections of EAE mice.
ATG-3⬘, antisense, 5⬘-TCTTGGCAGTCAGGTCGTTC-3⬘; The CNS leukocyte infiltration was also quantified by flow
and ␤-actin sense, 5⬘-GGCTGTATTCCCCTCCATCG-3⬘, cytometry analysis at day 17 postimmunization. The results
antisense, 5⬘-CCAGTTGGTAACAATGCCATGTT-3⬘. again confirmed that both the CD45⫹ infiltrates (Fig. 2, B, top

28658 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 34 • AUGUST 17, 2012
 VPA Ameliorates EAE Pathogenesis

FIGURE 1. VPA alleviates clinical symptoms of EAE. A–C, EAE was induced in female C57/B6 mice by immunization with MOG35–55. VPA was given once daily
starting from day 3 postimmunization via intraperitoneal injection (A) or oral administration (B) or starting from day 12 by oral administration (C) until the end
of the experiment, and clinical scores were recorded every day. Control groups were given oral or intraperitoneal injection of saline. The data represent the
means ⫾ S.E. (n ⫽ 10). ###, p ⬍ 0.001 (two-way ANOVA test). *, p ⬍ 0.05; **, p ⬍ 0.01; ***, p ⬍ 0.001 versus vehicle control (Mann-Whitney U test). D and
E, hematoxylin and eosin staining (D) and Luxol fast blue staining (E) of paraffin sections of spinal cords isolated from naive, vehicle, or VPA-treated (300 mg/kg,
orally, starting from day 3) EAE mice on day 17 after immunization. The boxed areas in the top rows are presented enlarged in the bottom row. F and G, number
of CNS infiltrates in the hematoxylin- and eosin-stained sections and the area of demyelination in the Luxol fast blue stained sections are quantified. Four
animals from each group were sacrificed, and 15 sections of the spinal cord of each animal were analyzed. ***, p ⬍ 0.001, versus naive control; ###, p ⬍ 0.001,
versus vehicle control (Student’s t test).

FIGURE 2. VPA reduces CNS infiltration of pathogenic T cells. A, frozen sections were prepared from spinal cords isolated from vehicle or VPA-treated EAE
mice at day 17 postimmunization, and CD45⫹ cells and CD4⫹ T cells were visualized with antibody staining. DAPI was used to stain the nuclei. For three
mice/group, six serial sections were stained, and representative sections are presented. The boxed areas in the first and third rows are presented enlarged in the
second and fourth rows, respectively. B–E, CNS infiltrates were isolated with 37/70% Percoll from naive and MOG-EAE mice treated with vehicle or VPA (300
mg/kg, orally, starting from day 3) at day 17 postimmunization and the number of CD45⫹ infiltrates (B, top panel, and C), CD4⫹ T cells (B, top panel, and D), and
TH-1 and TH-17 cells (B, bottom panel, and E) were analyzed by flow cytometry. The data represent the means ⫾ S.E. (n ⫽ 6). *, p ⬍ 0.05; **, p ⬍ 0.01 versus naive
control; #, p ⬍ 0.05, versus vehicle control (Student’s t test).

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VPA Ameliorates EAE Pathogenesis

FIGURE 3. VPA reduces periphery T cell population in EAE mice. A and B, naive and EAE mice were treated with VPA (300 mg/kg, orally, starting from day 3)
or vehicle. The body weight (B) and spleen weight (A) were measured at day 10. The data are the means ⫾ S.E. (n ⫽ 6). ***, p ⬍ 0.001, versus naive control; #, p ⬍
0.05, versus vehicle control (Student’s t test). C and D, leukocytes were isolated from the spleen (C) and circulating blood (D) of naive and EAE animals treated
with VPA (300 mg/kg, orally, starting from day 3) or vehicle control at day 10 postimmunization, and the percentage and number of CD4⫹ T cells, CD8⫹ T cells,
and B cells (B220⫹) were analyzed with surface staining and flow cytometry. TH-1 and TH-17 cells were analyzed by intracellular staining of IFN-␥ and IL-17a in
the CD4 gate. E–G, statistical analysis of C. H and I, statistical analysis of D. The data represent the means ⫾ S.E. (n ⫽ 6). *, p ⬍ 0.05; **, p ⬍ 0.01; ***, p ⬍ 0.001
versus naive control. #, p ⬍ 0.05; ##, p ⬍ 0.01; ###, p ⬍ 0.001 versus vehicle treatment group (Student’s t test).

row, and C) and CD4⫹ T cells (Fig. 2, B, top row, and D) accu- disease, but at day 10, there is no difference of body weight
mulated in the CNS of EAE mice were decreased after VPA between the naive and EAE animals (Fig. 3B).
treatment. The IL-17 producing TH-17 cells and the IFN-␥ pro- Surface staining of proper markers showed that the percent-
ducing TH-1 cells are the major pathogenic T effector cells in age and number of CD4⫹ T cells were dramatically increased in
EAE. Therefore we quantified the number of TH-1 and TH-17 EAE mice, whereas VPA treatment significantly reduced the
cells in the CNS infiltrates. After VPA treatment, the absolute percentage and the number of CD4⫹ T cells and CD8⫹ T cells
number of both TH-17 and TH-1 cells were significantly in the spleen. After EAE induction, the number of B cells in the
decreased (Fig. 2, B, bottom row, and E). Taken together, these spleen did increase, although the increase was not as dramatic
data indicate that VPA treatment significantly reduces EAE as T cells. So percent-wise, the frequency of B cells in the spleen
severity accompanied by decreased CNS inflammation and was not changed, and VPA did not significantly affect the B cells
demyelination. population (Fig. 3, C, top two rows, E, and F). With intracellular
VPA Reduces Peripheral CD4⫹ and CD8⫹ T Cells in EAE cytokine staining, we found that the number of TH-1 and TH-17
Mice—Expansion and differentiation of CD4⫹ T cells is cells in the CD4⫹ population was significantly reduced in the
believed to be a prerequisite of EAE pathogenesis (21). At day 10 spleen of VPA-treated EAE mice (Fig. 3, C, bottom row, and G).
postimmunization, we observed that the spleen of the EAE Similar results were obtained in the peripheral blood samples
mice were significantly larger than the naive controls even (Fig. 3, D, H, and I).
before the onset of the clinical symptoms, VPA treatment sig- VPA Blocks Proliferation and Induces Apoptosis in Activated
nificantly reduced the size of the spleen in EAE animals without T Cells—Our results showed that the frequency of T cells was
affecting the whole body weight (Fig. 3, A and B). The body greatly reduced in the periphery immune tissues upon VPA
weight of EAE mice usually drops quickly after the onset of the treatment. Based on these observations, we speculated that

28660 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 34 • AUGUST 17, 2012
 VPA Ameliorates EAE Pathogenesis

FIGURE 4. VPA blocks proliferation and induces apoptosis in activated T cells. A, naive mouse splenocytes were stimulated with 2 ␮g/ml anti-mouse CD3
and 2 ␮g/ml anti-mouse CD28 in the presence of VPA; cell proliferation was assessed with CFSE staining. B–D, representative FACS plots of apoptosis (annexin
V positive and PI negative) in total splenocytes (B), CD4⫹ (C), or CD8⫹ (D) T cells treated with VPA. E–G, statistical analysis of apoptosis presented in B–D. The data
are from three independent experiments (means ⫾ S.E.). ***, p ⬍ 0.001, versus vehicle control (two-way ANOVA). H, naive CD4⫹ T cells were induced to
differentiate into TH-1 or TH-17 cells in the presence of VPA. I, apoptosis of TH-1 and TH-17 cells induced by VPA (1 mM). The data are the means ⫾ S.E. of three
independent experiments. *, p ⬍ 0.05; **, p ⬍ 0.01; ***, p ⬍ 0.001 versus vehicle control (Student’s t test).

VPA might influence the proliferation, survival, or apoptosis of TH-17 cells for 72 h, and then VPA (1 mM) was added for
T cells. Splenocytes isolated from naive mice were activated another 48 h. VPA significantly increased the percentage of
with anti-mouse CD3 and anti-mouse CD28 in the presence of apoptotic cells in both TH-1 (IFN-␥⫹) and TH-17 (IL-17a⫹)
VPA for 48 h. The proliferation of T cells in vitro was deter- populations (Fig. 4I).
mined by the CFSE method. As shown in Fig. 4A, VPA dose-de- VPA Induces T Cell Apoptosis via Caspase Pathway—
pendently suppressed the T cell antigen-stimulated cell Caspase-dependent cascade is one of the major pathways that
proliferation. regulate cell apoptosis (23). We found the mRNA levels of
Next, early apoptosis in splenocytes and subpopulations was caspase-3 and 8 in the splenocytes isolated from EAE mice
measured with annexin V (positive) and PI (negative) staining treated with VPA were significantly up-regulated compared
after the treatment of VPA (0.3 and 1 mM) for various durations with the vehicle-treated ones (Fig. 5A). More interestingly, the
(24 –72 h). Activated splenocytes underwent apoptosis in a mRNA levels of caspases-3, -8, and -9 were only found to be
time-dependent fashion (Fig. 4, B and E) as previously reported significantly up-regulated in the T cell population after VPA
(14, 22). VPA enhanced apoptosis in splenocytes in a dose-de- treatment (Fig. 5, B and C). Caspases are cysteine proteases
pendent way (Fig. 4, B and E). Subpopulation analysis with cell critically involved in apoptosis. The caspases convey the apo-
surface markers revealed that VPA enhanced apoptosis in both ptotic signal in a proteolytic cascade, with the upstream
the CD4⫹ and CD8⫹ T cells after activation in vitro (Fig. 4, C, D, caspases (such as caspase-8 and -9) cleaving and activating the
F, and G). downstream ones, such as caspase-3, which lead to apoptosis by
Considering that TH-17 and TH-1 cells are the major patho- direct degradation of cellular targets (24). We then measured
genic T effector cells in EAE, we tested whether VPA affect their the protein level of caspase-3 in the splenocytes from EAE mice
differentiation and apoptosis in vitro. Naive CD4⫹ T cells were treated with VPA or vehicle. In the VPA-treated group, the
induced to differentiate into TH-1 or TH-17 cells by supplemen- acetylation level of H3 was significantly increased. The protein
tation with differentiation factors in the presence of VPA. The levels of both the pro-caspase-3 and cleaved caspase-3 (active
cells were harvested 72 h later, and intracellular staining for form) were also significantly higher in the VPA treatment
IFN-␥ or IL-17a was performed. Upon FACS analysis, VPA was group (Fig. 5, D and E).
found to significantly block the differentiation of both the TH-1 Next we tested the effect of VPA on MOG-specific T cells.
and TH-17 cells in vitro (Fig. 4H), and TH-17 differentiation Splenocytes were isolated from naive or EAE mice at day 10
seems more susceptible to VPA treatment. To assess the apo- postimmunization and reactivated with MOG (20 ␮g/ml) in
ptosis-inducing effect of VPA in TH-1 and TH-17 cells, naive vitro in the presence of VPA (1 mM) or the combination of VPA
CD4⫹ T cells were allowed to differentiate into TH-1 or and the pan-caspase inhibitor Z-VAD-FMK (10 ␮M) (25), and

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VPA Ameliorates EAE Pathogenesis

FIGURE 5. VPA induces T cell apoptosis via caspase pathway. A–C, total splenocytes (A), T cells (B), and non-T cells (C) were isolated from EAE mice
treated with VPA (300 mg/kg, orally, starting from day 3) or vehicle at day 10 postimmunization, and the expression levels of caspases-1, -3, -6, -8, and
-9 were analyzed with quantitative PCR. The data represent the means ⫾ S.E. (n ⫽ 6). *, p ⬍ 0.05; **, p ⬍ 0.01; ***, p ⬍ 0.001 versus vehicle control
(Student’s t test). D, splenocytes were isolated from EAE mice treated with VPA (300 mg/kg, orally, starting from day 3) or vehicle at day 10 postimmu-
nization, and the protein levels of pro-caspase-3, cleaved (activated) caspase-3, and acetylated histone H3 were analyzed with Western blot. The image
presented is representative of three independent experiments, and three mice from each group were analyzed. E, statistical analysis of the protein levels
presented in D. The data are the means ⫾ S.E. *, p ⬍ 0.05 versus vehicle control (Student’s t test). F and G, representative FACS plots of apoptosis in MOG
specific CD4⫹ (F) or CD8⫹ (G) T cells isolated from naive and EAE mice at day 10 treated with VPA (1 mM) or the combination of VPA (1 mM) and
Z-VAD-FMK (10 ␮M). H and I, statistical analysis of F and G. The data are from three independent experiments, each performed in triplicate (means ⫾ S.E.).
***, p ⬍ 0.001 (two-way ANOVA).

apoptosis in the CD4⫹ and CD8⫹ T subpopulations were mea- and B). As observed in mouse T cells, VPA dose-dependently
sured with FACS (Fig. 5, F and G). T cells from naive animals enhanced apoptosis in normal human CD4⫹ and CD8⫹ T cells
displayed a moderate level of apoptosis (Fig. 5, H and I, open in vitro (Fig. 6, C and D), and the effect of VPA was significantly
circles), whereas the T cells from EAE mice showed very low inhibited by the pan-caspase inhibitor, Z-VAD-FMK (Fig. 6, E
levels of apoptosis (closed squares). VPA treatment greatly pro- and F).
moted apoptosis in both MOG specificity CD4⫹ and CD8⫹ T Next, we tested VPA on the PBMCs isolated from three MS
cells (open triangles), and such an effect was significantly inhib- patients (Table 1). These cells were stimulated with MOG (20
ited by Z-VAD-FMK (closed triangles). Taken together, these ␮g/ml) in the presence of VPA (1 mM) or the combination of
data indicate that VPA induces T cell apoptosis via the caspase VPA (1 mM) and Z-VAD-FMK (20 ␮M). Compared with the T
pathway. cells from the healthy subjects, the cells isolated from the MS
VPA Promotes Human T Cell Apoptosis—It would be inter- patients showed minimal apoptosis in the first 48 h of in vitro
esting to know whether the apoptosis-inducing effect of VPA in culture (Fig. 7). VPA enhanced the apoptosis of both CD4⫹ and
mouse T cells could also be applied to human T cells. Human CD8⫹ T cells isolated from MS patients (Fig. 7) to a level similar
PBMCs were isolated from healthy subjects and stimulated to that of the spontaneous apoptosis observed in T cells isolated
with anti-human CD3 and anti-human CD28 in the presence of from healthy subjects (Fig. 6). This effect was significantly
VPA (0.3 or 1 mM) or the combination of VPA (1 mM) and inhibited by Z-VAD-FMK (Fig. 7). These data indicate that
Z-VAD-FMK (20 ␮M), and the apoptosis in the CD4⫹ and VPA also induces apoptosis in human T cells via the caspase
CD8⫹ T subpopulations was measured with FACS (Fig. 6, A pathway.

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 VPA Ameliorates EAE Pathogenesis

FIGURE 6. VPA promotes apoptosis in T cells from healthy human. A and B, representative FACS plots of apoptosis (annexin V positive and PI negative) in
healthy human CD4⫹ (A) or CD8⫹ (B) T cells treated with VPA (0.3 mM and 1 mM) or the combination of VPA (1 mM) and Z-VAD-FMK (20 ␮M) for 24, 48, or 72 h.
C and D, statistical analysis of VPA-induced apoptosis in CD4⫹ or CD8⫹ T cells presented in the top three rows in A and B. E and F, statistical analysis of the effect
of Z-VAD-FMK on VPA-induced apoptosis presented in the bottom row in A and B. The data are from three independent experiments (means ⫾ S.E.). ***, p ⬍
0.001 (two-way ANOVA).

TABLE 1 demyelination, and the induction of apoptosis in autoreactive T

Characteristics of patients cells might be one of the mechanisms by which VPA exerts its
Sample size is the total number of subjects. Age is presented in years ⫾S.E. Sex is therapeutic effect. Peripheral CD4⫹ T cells are maintained in a
presented as the total number (with percentage of group in parentheses). The
expanded Disability Status Scale (EDSS) score is presented as the mean ⫾ S.E. homeostatic balance between their production and elimination
Relapsing (30). Upon antigen stimulation, naive CD4⫹ T cells proliferate
Remitting MS
and differentiate into effector T cells to regulate immune
Sample size 3 responses. Then several distinct mechanisms, including cell
Age 46.7 ⫾ 5.0
Sex
inactivation, activation-induced cell death, and activated T cell-
Female 2 (67%) autonomous death, will lead to the reduction in the number and
Male 1 (33%) activity of effector T cells and thus the immune responses. Fail-
1.7 ⫾ 0.3
EDSS score
ure of CD4⫹ T cell death may prolong the immune responses
and result in disturbed T cell homeostasis and autoimmune
DISCUSSION diseases including MS (2). Recently, CD8⫹ T cells are also
Disregulation of cell survival and death can cause many dis- implicated in the pathogenesis of EAE. The CD8⫹ T lympho-
eases, including cancer and autoimmune diseases. Inducing cell cytes detected in MS lesions demonstrate characteristics of
apoptosis, a major strategy used in cancer therapy, might also activated and clonally expanded cells, supporting the notion
be useful in treating autoimmune disorders. In recent years, that these cells actively contribute to the observed injury (7).
many efforts have been made to develop HDACis as a cancer Our results demonstrated that VPA enhances apoptosis in anti-
treatment (26, 27). Currently, two HDACis, vorinostat and CD3/CD28 activated or MOG-restimulated CD4⫹ and CD8⫹
romidepsin, are approved to treat cutaneous T cell lymphoma T cells, but not in B cell population, indicating that the main
(17, 18), and a number of HDACis are at various stages of clin- effect of VPA is to restore T cell homeostasis in EAE mice.
ical study. Among them, VPA, the well known anti-psychotic Two major pathways have been implicated in regulating the
drug, is under clinical investigation to treat breast cancer, ovar- death of T cells. One is the mitochondria-mediated pathway
ian cancer, various form of leukemia, and many other malig- modulated by the Bcl-2 protein family, which is composed of
nancies (listed on the National Institutes of Health Clinical Tri- pro- and antiapoptotic members. In Bcl-2-deficient mice, T
als website). The exact mechanisms by which these compounds cells are more susceptible to death (31, 32), whereas in Bcl2-
may work are still unclear, but epigenetic pathways that even- transgenic mice, lymphocytes are more resistant to apoptotic
tually lead to cell growth arrest, differentiation, or apoptosis stimuli, and autoimmune symptoms have been reported (33).
have been proposed (29). Such epigenetic modulatory and pro- The other pathway is through the activation of caspase cascade
apoptotic activity also lead to the investigation of HDACis in (34, 35), which is typically induced by cell surface receptors
the inflammatory diseases (20). such as TNF, TRAIL receptors, and Fas, which are death factors
Herein, we demonstrated that VPA effectively reduces EAE for activated CD4⫹ T cells. In our study, VPA was found to
severity accompanied by decreased CNS inflammation and significantly and specifically enhance the mRNA expression of

AUGUST 17, 2012 • VOLUME 287 • NUMBER 34 JOURNAL OF BIOLOGICAL CHEMISTRY 28663
VPA Ameliorates EAE Pathogenesis

FIGURE 7. VPA promotes apoptosis in T cells from MS patients. A and B, representative FACS plots of apoptosis (annexin V positive and PI negative) in MS
patient CD4⫹ (A) or CD8⫹ (B) T cells treated with VPA (1 mM) or the combination of VPA (1 mM) and Z-VAD-FMK (20 ␮M) for 24, 48, or 72 h. C and D, statistical
analysis of A and B. The data are from three independent experiments (means ⫾ S.E.). ***, p ⬍ 0.001 (two-way ANOVA).

caspases-3, -8, and -9 in T cells. Caspase-3 is a downstream 3. Massacesi, L. (2002) Compartmentalization of the immune response in
caspase that is cleaved and activated by upstream caspases such the central nervous system and natural history of multiple sclerosis. Im-
plications for therapy. Clin. Neurol. Neurosurg. 104, 177–181
as caspase-8 and 9 and that leads to apoptosis by direct degra-
4. Camelo, S., Iglesias, A. H., Hwang, D., Due, B., Ryu, H., Smith, K., Gray,
dation of cellular targets (24). We found the protein levels of S. G., Imitola, J., Duran, G., Assaf, B., Langley, B., Khoury, S. J., Stephano-
both the pro-caspase-3 and cleaved caspase-3 (active form) poulos, G., De Girolami, U., Ratan, R. R., Ferrante, R. J., and Dangond, F.
were also significantly higher in the splenocytes of VPA-treated (2005) Transcriptional therapy with the histone deacetylase inhibitor tri-
mice. The fact that the pan-caspase inhibitor Z-VAD-FMK chostatin A ameliorates experimental autoimmune encephalomyelitis.
effectively blocked VPA-induced apoptosis in T cells further J. Neuroimmunol. 164, 10 –21
5. Jäger, A., Dardalhon, V., Sobel, R. A., Bettelli, E., and Kuchroo, V. K. (2009)
confirmed our hypothesis that VPA enhances T cell death via
Th1, Th17, and Th9 effector cells induce experimental autoimmune en-
the caspase pathway. cephalomyelitis with different pathological phenotypes. J. Immunol. 183,
Other mechanisms have also been proposed to underlie the 7169 –7177
beneficial effects of HDACis in autoimmunity. IL-2 is a cyto- 6. York, N. R., Mendoza, J. P., Ortega, S. B., Benagh, A., Tyler, A. F., Firan, M.,
kine essential for immune responses and T cell homeostasis and Karandikar, N. J. (2010) Immune regulatory CNS-reactive CD8⫹T
(36). HDACi trichostatin A and FR235222 have been shown to cells in experimental autoimmune encephalomyelitis. J. Autoimmun. 35,
33– 44
inhibit IL-2 gene expression and to possess immunosuppressive 7. Mars, L. T., Saikali, P., Liblau, R. S., and Arbour, N. (2011) Contribution of
activity in vivo (37, 38). Trichostatin A has also been shown to CD8 T lymphocytes to the immuno-pathogenesis of multiple sclerosis
up-regulate antioxidant, anti-excitotoxicity, and pro-neuronal and its animal models. Biochim. Biophys. Acta 1812, 151–161
growth and differentiation mRNAs and protect neurons from 8. Wahl, S. M., Wen, J., and Moutsopoulos, N. (2006) TGF-␤. A mobile
apoptosis in EAE (4). VPA has been reported to remarkably purveyor of immune privilege. Immunol. Rev. 213, 213–227
attenuate MCAO-induced blood-brain barrier disruption and 9. Anderson, A. C., Reddy, J., Nazareno, R., Sobel, R. A., Nicholson, L. B., and
Kuchroo, V. K. (2004) IL-10 plays an important role in the homeostatic
brain edema via HDAC inhibition-mediated suppression of regulation of the autoreactive repertoire in naive mice. J. Immunol. 173,
NF-␬B activation, MMP-9 induction, and tight junction degra- 828 – 834
dation (39). 10. Fan, H. C., Ren, X. R., Yu, J. Z., Guo, M. F., Ji, N., Sun, Y. S., Liang, L. Y., and
With their combined neuroprotective, neurotrophic, and Ma, C. G. (2009) [Suppression of murine EAE by triptolide is related to
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11. Huss, D. J., Winger, R. C., Cox, G. M., Guerau-de-Arellano, M., Yang, Y.,
long history of clinical use in psychiatry and neurology and our Racke, M. K., and Lovett-Racke, A. E. (2011) TGF-␤ signaling via Smad4
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12. Brunner, T., Wasem, C., Torgler, R., Cima, I., Jakob, S., and Corazza, N.
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