Preliminary report

Immunomodulatory effects and improved prognosis of experimental autoimmune

encephalomyelitis after O-tetradecanoyl-genistein treatment
Sandra B.R. Castro
a
, Celso O.R. Junior
b
, Caio C.S. Alves
a
, Alyria T. Dias
a
, Lvia L. Alves
a
, Luciano Mazzoccoli
a
,
Felipe P. Mesquita
a
, Nathlia S.V. Figueiredo
a
, Maria A. Juliano
c
, Maria Christina M.N. Castaon
d
,
Jacy Gameiro
a
, Mauro V. Almeida
b
,
Henrique C. Teixeira
a, 1
, Ana Paula Ferreira
a,

, 1
a
Department of Parasitology, Microbiology and Immunology, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Brazil
b
Department of Chemistry, Federal University of Juiz de Fora, Juiz de Fora, Brazil
c
Department of Biophysics, Federal University of So Paulo, So Paulo, Brazil
d
Department of Morphology, Federal University of Juiz de Fora, Juiz de Fora, Brazil
a b s t r a c t a r t i c l e i n f o
Article history:
Received 20 August 2011
Received in revised form 29 December 2011
Accepted 30 December 2011
Available online 13 January 2012
Keywords:
EAE
Multiple sclerosis
Genistein
IL-17
Foxp3
IFN-
Experimental autoimmune encephalomyelitis (EAE) is a murine autoimmune disease used to study multiple
sclerosis (MS), a human inammatory demyelinating disease of the central nervous system. Genistein, an
isoavonoid phytoestrogenic compound found in soy, is known to reverse clinical signs of EAE. Although
genistein has some potential in clinical application, it has some disadvantages related to its chemical struc-
ture, such as rapid in vivo metabolism and a fast decline in serum after oral administration. The present
work investigates the treatment of EAE by using 7-O-tetradecanoyl-genistein (TDG), a more lipophilic analog
of genistein obtained by esterication. The clinical course of EAE was investigated in C57Bl/6 mice immu-
nized with myelin oligodendrocyte glycoprotein peptide (MOG)
3555
in complete Freund's adjuvant supple-
mented with Mycobacterium tuberculosis H37RA. After 14 days of MOG immunization, mice were treated
with TDG for seven days. Numbers of IL-17-producing cells and Foxp3 by CD4
+
T cells and CTLA-4 expression
by CD3
+
T cells from brain were determined by ow cytometry. Levels of IL-6, IFN- and IL-10 were evalu-
ated by ELISA. Brain sections were stained by hematoxylin and eosin method. The data obtained indicate that
TDG treatment ameliorates the clinical signs of EAE, which correlates with a decrease of IL-17-producing cells
and an increase in Foxp3
+
CD4
+
cells in the brain. TDG is also shown to enhance IL-10 production and CTLA-4
expression and to reduce IFN- and IL-6. Altogether, these ndings suggest an immunomodulatory therapeu-
tic role for TDG in EAE and multiple sclerosis.
2012 Elsevier B.V. All rights reserved.
1. Introduction
Multiple sclerosis (MS) is a chronic inammatory demyelinating
disease that affects the central nervous system (CNS) [1,2]. Experi-
mental autoimmune encephalomyelitis (EAE) is a model for the
study of MS. Cytokines produced by T-helper 1 (Th1) cells, such as
IFN-, were considered primarily responsible for the induction of
the inammatory process in CNS during the development of MS and
EAE [3,4]. The importance of T-helper 17 (Th17) cells in this process
has been described [57]. Th17 can disrupt the bloodbrain barrier
by the action of IL-17 [8]. Studies suggest that blockage or neutraliza-
tion of IL-17 could be a therapeutic strategy to improve the clinical
signs of EAE, highlighting the importance of IL-17 in the development
of EAE [6,8]. However, regulatory T cells (Tregs) have been also
reported in studies of EAE as an important cell population which pre-
vents and restrains the EAE development, mainly because of IL-10
production [9,10].
Genistein is an isoavonoid derived from soy that can affect many
different cellular mechanisms such as the inhibition of tyrosine ki-
nases. It increases the activity of antioxidant enzymes and decreases
the production of pro-inammatory molecules by an inhibitory effect
on the classical NF-B activation factor, known to play a critical role in
inammation, immune modulation and cell proliferation [1113].
Genistein has been shown to down-modulate the pro-inammatory
cytokines such as IFN-, IL-12 and TNF-, and to reverse clinical
signs of EAE [14]. Although genistein has some potential clinical
application, it has some disadvantages related to its chemical struc-
ture, such as rapid in vivo metabolism and a fast decline in serum
after oral administration [15,16]. The esterication in vivo of genis-
tein could be responsible for the increase of biological activity [17].
International Immunopharmacology 12 (2012) 465470
Corresponding author at: Department of Parasitology, Microbiology and Immunology,
Institute of Biological Sciences, Federal University of Juiz de Fora, 36036-900, Juiz de Fora,
Minas Gerais, Brazil. Tel./fax: +55 32 21023214.
E-mail address: ana.paula@ufjf.edu.br (A.P. Ferreira).
1
A.P.F. and H.C.T. share last authorship.
1567-5769/$ see front matter 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.intimp.2011.12.025
Contents lists available at SciVerse ScienceDirect
International Immunopharmacology
j our nal homepage: www. el sevi er . com/ l ocat e/ i nt i mp
A correlation between hydrophobic character, membrane permeabil-
ity and biological activity is frequently observed [18,19]. In this work,
a novel analog of genistein, the 7-O-tetradecanoyl-genistein (TDG),
was developed using esterication in vitro to bypass the in vivo pro-
cessing, which favors biological activity and increased lipid solubility.
The aim of this study was to evaluate the effect of TDG treatment on
IL-17-producers and Foxp3 expression by CD4
+
T cells. Levels of IL-6,
IFN- and IL-10 in brain homogenates after TDG treatment were also
investigated.
2. Materials and methods
2.1. Chemical compounds
7-O-tetradecanoyl-genistein (TDG) was synthesized as shown in
Fig. 1. The genistein ester can be easily obtained, in high yield, by
the esterication reaction of genistein with tetradecanoic acid in the
presence of dicyclohexylcarbodiimide (DCC) and dimethylaminopyr-
idine (DMAP) in dichloromethane. Genistein (log (P): 1.4) (Sigma, St.
Louis, MO, USA) and its analog TDG (log (P): 6.3) were solubilized in
the DMSO (Sigma), never exceeding 0.1% (v/v), and diluted in sterile
phosphate buffered saline (PBS). The partition coefcient P (log (P))
of genistein and TDG represents the hydrophobic properties of these
compounds.
2.2. Animals
Female C57Bl/6 mice 46 weeks old were obtained from the
animal care facilities of the Federal University of Juiz de Fora (UFJF)
and maintained in microisolator cages. All procedures were in
accordance with the principles of the Brazilian Code for the Use of
Laboratory Animals. Mice were used between 8 and 10 weeks of age
(n=17 per group). All protocols involving mice handling were
approved by the committee on the use of laboratory animals from
UFJF (Protocol no. 026/2008).
2.3. EAE induction and TDG treatment
Three groups of 17 mice were subcutaneously (s.c.) immunized at
the tail base with 100 g of myelin oligodendrocyte glycoprotein
peptide (MOG)
3555
, synthesized in the laboratory of Biophysics,
Federal University of So Paulo, emulsied v/v in complete Freund's
adjuvant (Sigma Chemical Co., Saint Louis, USA) and supplemented
with 400 g of attenuated Mycobacterium tuberculosis H37RA (Difco,
Detroit, USA). Pertussis toxin 300 ng/animal (Sigma Chemical Co.,
Saint Louis, USA) was injected intraperitoneally (i.p.) on the day of
immunization and 48 h later. One of these immunized groups was
treated with TDG (TDG group) at a dose of 200 mg/kg s.c daily for a
total of seven doses. This treatment was initiated at day 14 post-
immunization (p.i.) [14]. A second immunized group received no
treatment (NT-EAE group). Genistein was used as a reference
compound on the third immunized group (genistein group) at a
dose of 200 mg/kg s.c, because of its proven effects on EAE [14]. A
non-immunized control group (CT group) was s.c. injected with
dimethyl sulfoxide (DMSO, Sigma Chemical Co., Saint Louis, USA)
0.1%. Animals were monitored daily and neurological impairment
was quantied.
2.4. Clinical assessment
Mice were weighed and the clinical signs of EAE observed daily for
up to 21 days after immunization. The clinical status was assessed
individually according to Table 1 [14]. The nal clinical score was
obtained adding all individual scores assessed.
2.5. Isolation of brain mononuclear cells
Twenty-one days after infection, groups of mice (n=8) were
euthanized under anesthesia (i.p) and perfused through the left ven-
tricle with phosphate saline buffer (PBS). Brains were macerated and
ltrated using 70 m cell strainer (BD Biosciences, Bedford, USA) in
RPMI medium 1640 (Gibco, Grand Island, USA) with 10% fetal bovine
serum (FBS) (Gibco, Grand Island, USA). Dispersed brain cells were
incubatedin RPMI containing 2 mg of collagenase D(Roche, Mannheim,
Germany) at 37 C for 45 min. Brain mononuclear cells were collected
after Percoll density gradient centrifugation [20] and washed with
staining buffer (PBS, 1% FBS, 0.1% sodium azide). The cell pellet was
suspended in ACK solution, centrifuged at 350g for 5 min, and
suspended in staining buffer for cytometry analysis.
2.6. Cytometry analysis
Brain cells were incubated with anti-mouse CD3-PerCP, anti-mouse
CTLA-4-PE or anti-mouse CD4-PerCP (BD Biosciences Pharmingen, San
Diego, USA). After 30 min at 4 C, cells were washed in staining buffer
and permeabilized with BD Pharmingen Fix/Perm solution, washed in
BD Pharmingen Perm/Wash Buffer, and then stained with anti-mouse
IL-10-PE, anti-mouse IL-17-Alexa Fluor 488, anti-mouse transcription
factor forkhead box P3 (Foxp3)-Alexa Fluor 488 (BD Biosciences
Pharmingen, San Diego, USA) and analyzed using a FACScalibur ow
cytometer and CellQuest software (Becton Dickinson, San Diego, USA).
2.7. Cytokine production
Brains from5 mice per group were removed and hemispheres were
homogenized (100 mg/ml) in extraction solution containing 0.4 M
NaCl, 0.05% Tween 20 (Merck & Co., Inc., Whitehouse Station, USA),
0.5%bovine serumalbumin (BSA), 0.1 Mphenylmethylsulfonyl uoride
(PMSF), 0.1 M benzethonium chloride, 10 mM ethylenediaminetetraa-
cetic acid (EDTA) and 20 kIU/ml aprotinin (Sigma Chemical Co., Saint
Louis, USA). The homogenate was centrifuged at 2000g for 15 min
at 4 C and supernatants were collected to determine the concentration
of IL-6, IL-17 and IFN-. Cytokine production was assayed by ELISA
using commercially available antibodies according to the manufac-
turer's instructions (BD Biosciences Pharmingen, San Diego, USA).
2.8. Histological analysis
To assess the degree of CNS inammation, 4 mice per group were
euthanized under anesthesia on day 21 post-immunization. The brain
was removed quickly. A portion of brain sample was xed in 10%
neutral buffered formalin and then cut into 5-m-thick sections.
Tissues were stained with hematoxylin and eosin (H&E) to assess
tissue damage and inammation.
Fig. 1. Preparation of 7-O-tetradecanoyl-genistein (2) by esterication reaction of genistein (1).
466 S.B.R. Castro et al. / International Immunopharmacology 12 (2012) 465470
2.9. Statistical analysis
Results represent at least two independent experiments and are
presented as the meanSEM. All data were analyzed using Kruskall
Wallis test (GraphPad Prism5.00), and the differences were considered
signicant at pb0.05.
3. Results
3.1. TDG treatment improves clinical signs of EAE
The clinical course of EAE was investigated in mice immunized
with MOG
3555
peptide and s.c. treated with TDG at 200 mg/kg for
seven days. EAE severity was recorded daily using a clinical score
scale (Fig. 2). The immunized mice presented signs of EAE such as
loss of muscle tone in tail around 1112 days after immunization.
On day 14 all immunized mice showed clinical scores including at
least tail paralysis (score=2). At 18th day post-immunization, the
clinical score (2.40.24) of the TDG treated mice was lower than
that observed in the NT-EAE group (score=40.45). Clinical
evidence of disease in the TDG-treated group peaked at day 15 after
immunization maintaining a low clinical score, while in the NT-EAE
group it peaked at day 17. These clinical score improvements after
TDG treatment were similar to those observed in the genistein
group (Fig. 2).
3.2. Cells and cytokines involved in EAE were modulated by TDG
treatment
The number of mononuclear cells isolated from the brains was
obtained to CT group (0.210
5
), NT-EAE (710
5
), TDG (2.210
5
)
and genistein (1.510
5
). IL-17-producing Th17 cells play an impor-
tant role in development of EAE [3,8]. To determine the presence of
cells producing IL-17, the brain cells were isolated and stained with
anti-mouse CD4 and anti-mouse IL-17. The percentage of IL-17-
producing cells was higher in the NT-EAE group in comparison to all
other groups. A lower number of IL-17-producing cells in the brain
were observed in the TDG group (Fig. 3A).
IL-6 is involved in the differentiation of Th17 [21]. The concentra-
tion of IL-6 (Fig. 3G) was measured in brain supernatants at day 21
after MOG
3555
immunization. Higher levels of IL-17, IFN- and IL-6
(Fig. 3EG) were observed in the NT-EAE group in relation to the CT
group. After TDG treatment, levels of IL-17, IFN- and IL-6 were
lower in comparison to the NT-EAE group (Fig. 3EG). These results
indicate an inuence of TDG treatment in down-modulation of the
pro-inammatory cytokines, IL-17, IFN- and IL-6.
Foxp3 is essential to establish functional Treg cells [22,23]. Treg
cells inhibit autoimmunity and protect against tissue lesions [22,24].
In this study, TDG treatment increased Foxp3
+
CD4
+
cells in levels
similar to the CT group and more than NT-EAE. This up-regulation
was also observed in genistein treated mice (Fig. 3B). In addition,
TDG treatment increased the number of Foxp3
+
CD4
+
T cells in
spleen and lymph node cells, in relation to either the NT-EAE group
or the genistein group (data not shown).
CTLA-4 is a potent down-regulator of T cell immune responses.
Expression of CTLA-4 was associated with Foxp3
+
Treg function
[25,26]. A lower percentage of CTLA-4
+
CD3
+
cells were observed in
the NT-EAE group (Fig. 3C). Mice treated with TDG had percentage
of CTLA-4
+
CD3
+
cells similar to the CT group, and high percentage
in comparison to the genistein group (Fig. 3C).
IL-10 is a regulatory cytokine that plays a critical role in control-
ling autoimmune diseases such as EAE [10]. The TDG treatment
increased the percentage of brain CD4
+
T cells producing IL-10 in
relation to the NT-EAE group (Fig. 3D). This increase was observed
also in the genistein group as compared with the NT-EAE group
(Fig. 3D).
3.3. TDG treatment reduces cellular inltration at brain of EAE mice
In the histological analysis of slides of tissue from the brain,
stained with H&E, was rst observed that the induction of EAE led
to the formation of a meningeal inammatory cell inltrate in the
brain of the induced animals (NT-EAE group) compared to the nega-
tive non-induced control group (CT group). The histological sections
of the brain of the CT group (Fig. 4A,B,C) were considered normal,
that is, without the presence of inammatory cell inltrate. In con-
trast all animals in group NT-EAE (Fig. 4D,E,F) showed meningeal
inammatory cell inltrate in the brain. In the analysis of histological
sections of brain of animals treated with the TDG (Fig. 4G,H,I), as well
as in animals treated with the genistein (Fig. 4J,K,L) meningeal
inammatory inltrates were not observed. The general appearance
of the histological sections of brain of the TDG and of the genistein
group is similar to the CT group.
4. Discussion
TDG is a lipophilic analog of genistein obtained by its esterication
reaction. The lipophilic character can be calculated by log (P) value,
where compounds with higher log values (P) are more lipophilic.
This increase of lipophilic character of the TDG analog was obtained
by the introduction of a hydrocarbon chain in the genistein com-
pound by means of an esterication reaction. The main ndings of
Table 1
Clinical score assessment.
Part of the body Clinical signs Score
a
Tail No clinical signs 0
Loss of muscle tone in tail 1
Paralysis 2
Hind-limb No clinical signs 0
Weakness of one animal paw 1
Weakness of both animal paws 2
Paralysis of one animal paw 3
Paralysis of both animal paws 4
Front-limb No clinical signs 0
Weakness of any animal paw 1
Paralysis of any animal paw 2
Bladder Continence 0
Incontinence 1
a
Numerical score arbitrarily established.
Fig. 2. Clinical score of C57Bl/6 mice immunized with MOG
3555
peptide (n=17 per
group). The clinical signs were registered from day 0 to day 21 post-immunization.
7-O-tetradecanoyl-genistein (TDG) and genistein treatments started at day 14 after
immunization. NT-EAE = untreated control group. Genistein = genistein-treated
group. TDG = TDG-treated group. Each point represents meanSEM. *pb0.01 of
Genistein versus NT-EAE, and TDG versus NT-EAE.
467 S.B.R. Castro et al. / International Immunopharmacology 12 (2012) 465470
this study are that treatment with TDG ameliorates the EAE clinical
course and this correlates with reduction of IL-17-producing cells in
the brain, lower levels of IL-17, IL-6 and IFN-, and an increased
number of Foxp3
+
CD4
+
T cells and IL-10 in CNS in comparison to
the NT-EAE group.
To our knowledge this is the rst study to show the action of a
genistein analog (TDG) in ameliorating the course of EAE. EAE patho-
genesis is mainly attributed to Th17 and its cytokine IL-17 [8]. A
decreased number of IL-17-producing cells were observed in the
TDG and in genistein treated groups. Results presented by others
showed the involvement of IL-17 in disrupting the bloodbrain
barrier, an early and central event in EAE pathogenesis [8,27]. IL-17
acts by formation of reactive oxygen species (ROS) that down-
regulate tight junction proteins and activate the endothelial contrac-
tile machinery [8]. Moreover, IL-17-neutralizing antibodies prevented
the bloodbrain barrier disruption induced by IL-17 and ameliorated
the EAE course [28]. The development of EAE is signicantly inhibited
in IL-17 decient mice. These animals exhibited delayed disease
onset, reduced maximum severity scores, ameliorated histological
changes and early recovery [29].
In this work, IL-17 and IL-6 levels in the brain were reduced after
TDG treatment. IL-6 is an important cytokine involved in differentia-
tion of naive T cells to Th17 subsets [21,30,31]. The presence of IL-6 is
decisive to the differentiation of Th17 subsets [21,30,31]. IL-6 has
been shown to drive up-regulation of the transcription factor orphan
nuclear receptor (RORt) and consequent induction of IL-17 produc-
tion [21]. The IL-6 blockade by treatment with anti-IL-6R inhibits the
development of EAE and Th17 differentiation in inguinal lymph nodes
[31]. Thus, the reduction in IL-6 with TDG treatment may have
favored the decline in production of IL-17. IFN- was also inhibited
by TDG treatment. IFN- production by Th1 cells correlates with the
seriousness of EAE [3,32,33].Together with Th17 cells, Th1 cells are
considered critical cell population responsible for EAE development,
mainly through IFN- production [32,33].
TDG treatment increased the expression of transcription factor
Foxp3 in CD4
+
T cells. Foxp3 is essential to establish functional
Tregs [22,23]. The importance of Tregs in suppressing EAE is widely
recognized [3436]. A decrease of Foxp3-expressing cells has been
observed in the NT-EAE group. Studies have shown that pertussis
toxin used as an adjuvant in the induction of EAE with MOG peptide
Fig. 3. Expression of intracellular IL-17 (A), Foxp3 (B), IL-10 (D) in brain CD4 T and CTLA-4 in brain CD3 T cells (C) were determined by ow cytometry (n=8 mice/group) at day 21
post-immunization with MOG
3555
. Cells were stained with uorescence-conjugated antibodies to IL-17 (Alexa Fluor 488), Foxp3 (Alexa Fluor 488), IL-10 (PE), CD4 (PerCP), CTLA-4
(PE) and CD3 (PerCP). IL-17 (E), IFN- (F), and IL-6 (G) were measured by ELISA (n=5 mice/group) in brain homogenates at day 21 post-immunization with MOG
3555
. Bars
represent meanSEM. CT = non-immunized control. NT-EAE = untreated, immunized control. Genistein = immunized group treated with genistein. TDG = immunized
group treated with 7-O-tetradecanoyl-genistein. *pb0.05 versus genistein. **pb0.05 versus NT-EAE.
468 S.B.R. Castro et al. / International Immunopharmacology 12 (2012) 465470
promotes the reduction of Treg cells [37,38]. In addition, the presence
of IL-6 may inhibit the generation of Tregs in favor of Th17 in the
presence of TGF- [23,24].
Up-regulation of IL-10 was observed after TDG treatment, and was
associated with an increase in the percentage of Foxp3
+
CD4
+
cells.
IL-10 is produced primarily by Treg cells [9]. IL-10 has been shown
to be a critical cytokine that suppresses effector T-cell function
[9,10]. Previous studies have demonstrated that EAE clinical signs
are reduced by a IL-10-dependent mechanism [9,10,39]. In addition,
in this work, an increased expression of CTLA-4 was observed in
TDG-treated group. CTLA-4 is expressed by T lymphocytes and bind
to CD80 and CD86 molecules in antigen-presenting cells (APCs),
competing with the coactivation molecule CD28 [40]. CTLA-4 may
exert a key role in the suppressive function of Foxp3
+
Treg cells
[25]. Previous study has shown that increase in CTLA-4 expression
in lymphocytes controls the expansion of encephalitogenic cells in
EAE [41]. Therefore, we can hypothesize that the increased CTLA-4
expression in the TDG-treated group may favor the action of Treg
cells in controlling EAE. However, TDG and genistein act differently
in CTLA-4 expression and further studies are needed to assess this
different modulation.
Histological analysis of brain tissue, from animals belonging to the
experimental groups, demonstrated that the induction of EAE led to
the formation of inammatory cell inltrate in the brain of the NT-
EAE group in relation of the CT-group. Previous studies carrying out
histological analysis of the CNS tissues of animals with EAE often
showed the presence of meningeal inammatory cell inltrate in
the parenchyma of the brain and spinal cord of the animals
[36,4244]. The subarachnoid space is considered the starting
location for entry of cells in the CNS because of the constitutive
expression of chemokine receptors which would facilitate the entry
of inammatory cells, explaining the presence of meningeal inam-
matory cell inltrate in the CNS [4547].
The results obtained showed a reduction in cell inltration in the
CNS tissues in treatment with the TDG or the genistein, which could
explain the reduction in clinical signs of animals treated with the
TDG. Moreover, the results of this study support the hypothesis that
the worsening of clinical symptoms of EAE is correlated with the
inux of cells in the brain and spinal cord, leading to an increase in
the cells producing IL-17 and IFN-.
In conclusion, our ndings suggest that treatment with TDG, a
more lipophilic analog of genistein, improves the clinical signs of
EAE. This correlates with a decrease in IL-17-producing cells and an
increase in Foxp3
+
CD4
+
cells in the brain. TDG is also shown to
enhance IL-10 production and CTLA-4 expression, and to reduce
IFN- and IL-6. This study suggests a possible advantage of TDG use
for the treatment of EAE and multiple sclerosis. Further studies are
needed for a better understanding of the immunomodulation process
in EAE.
Acknowledgments
This work was supported in part by grants fromConselho Nacional de
Desenvolvimento Cientco e Tecnolgico (CNPq 481459/2009-0 and
303369/2009-4), Fundao de Amparo Pesquisa do Estado de Minas
Fig. 4. Brains from C57Bl/6 mice immunized with MOG
3555
peptide. The gures are representative of the histological analysis of each experimental group: CT = non-immunized
group (A, B, C), NT-EAE = immunized untreated group (D, E, F), TDG = immunized group treated with O-tetradecanoyl genistein (G, H, I), genistein=immunized group treated
with genistein (J, K, L), stained with H&E and captured at a magnication of 20, 40 and 10, bar scale=50 m. In histological sections of brains of the C57Bl/6 belonging to the
group NT-EAE the presence of meningeal inammatory inltrate is observed (arrows) absent in the CT group, without histological alterations. In the treatment with TDG or with
genistein inammatory inltrate was not observed, similar to that observed in the CT group.
469 S.B.R. Castro et al. / International Immunopharmacology 12 (2012) 465470
Gerais (FAPEMIG 01110/08 and PPM 0216/10) and Coordenao de
Aperfeioamento de Pessoal de Nvel Superior (CAPES).
References
[1] Ishizu T, Osoegawa M, Mei FJ, Kikuchi H, Tanaka M, Takakura Y, et al. Intrathecal
activation of the IL-17/IL-8 axis in opticospinal multiple sclerosis. Brain
2005;128:9881002.
[2] Steinman L. Multiple sclerosis: a two stage disease. Nat Immunol 2001;2:7624.
[3] Hedegaard CJ, Krakauer M, Bendtzen K, Lund H, Sellebjerg F, Nielsen CH. T helper
cell type 1 (Th1), Th2 and Th17 responses to myelin basic protein and disease
activity in multiple sclerosis. Immunology 2008;125:1619.
[4] Sospedra M, Martin R. Immunology of multiple sclerosis. Annu Rev Immunol
2005;23:683747.
[5] Kawanokuchi J, Shimizu K, Nitta A, Yamada K, Mizuno T, Takeuchi H, et al.
Production and functions of IL-17 in microglia. J Neuroimmunol 2008;194:5461.
[6] Hofstetter HH, Ibrahim SM, Koczan D, Kruse N, Weishaupt A, Toyka KV, et al.
Therapeutic efcacy of IL-17 neutralization in murine experimental autoimmune
encephalomyelitis. Cell Immunol 2005;237:12330.
[7] Kong W, Yen JH, Ganea D. Docosahexaenoic acid prevents dendritic cell
maturation, inhibits antigen-specic Th1/Th17 differentiation and suppresses
experimental autoimmune encephalomyelitis. Brain Behav Immun 2011;25:
87282.
[8] Huppert J, Closhen D, Croxford A, White R, Kulig P, Pietrowski E, et al. Cellular
mechanisms of IL-17-induced bloodbrain barrier disruption. FASEB J 2010;24:
102334.
[9] Spach KM, Nashold FE, Dittel BN, Hayes CE. IL-10 signaling is essential for 1,25-
dihydroxyvitamin D3-mediated inhibition of experimental autoimmune enceph-
alomyelitis. J Immunol 2006;177:60307.
[10] Cua DJ, Hutchins B, LaFace DM, Stohlman SA, Coffman RL. Central nervous system
expression of IL-10 Inhibits autoimmune encephalomyelitis. J Immunol
2001;166:6028.
[11] Gadgeel SM, Ali S, Philip AP, Wozniak A, Sarkar FH. Genistein enhances the effect
of epidermal growth factor receptor tyrosine kinase inhibitors and inhibits nucle-
ar factor kappa B in nonsmall cell lung cancer cell lines. Cancer 2009;115:
216576.
[12] Dijsselbloem N, Goriely S, Albarani V, Gerlo S, Francoz S, Marine JC, et al. A critical
role for p53 in the control of NF-kappa B-dependent gene expression in TLR-4-
stimulated dendritic cells exposed to genistein. J Immunol 2007;178:504857.
[13] Huang X, Chen S, Xu L, Liu Y, Deb DK, Platanias LC, et al. Genistein inhibits p38
map kinase activation, matrix metalloproteinase type 2, and cell invasion in
human prostate epithelial cells. Cancer Res 2005;65:34708.
[14] De Paula ML, Rodrigues DH, Teixeira HC, Barsante MM, Souza MA, Ferreira AP. Ge-
nistein down-modulates pro-inammatory cytokines and reverses clinical signs
of experimental autoimmune encephalomyelitis. Int Immunopharmacol 2008;8:
12917.
[15] Kwon SH, Kang MJ, Huh JS, Ha KW, Lee JR, Lee SK, et al. Comparison of oral bio-
availability of genistein and genistin in rats. Int J Pharm 2007;337:14854.
[16] Soucy NV, Parkinson DH, Sochaski MA, Borghoff SJ. Kinetics of genistein and its
conjugated metabolites in pregnant SpragueDawley rats following single and re-
peated genistein administration. Toxicol Sci 2006;90:23040.
[17] Lewis PT, Whl K, Hoikkala A, Mutikainen I, Meng QH, Adlercreutz H, et al. Syn-
thesis of antioxidant isoavone fatty acid esters. Tetrahedron 2000;56:780510.
[18] Corra TA, Reis EFC, Alves LL, Alves CCS, Castro SBR, Dias AT, et al. Preparation of
amino alcohols condensed with carbohydrates: evaluation of cytotoxicity and in-
hibitory effect on NO production. Chem Biol Drug Des 2010;76:4516.
[19] Polkowski K, Popiolkiewicz J, Krzeczynski P, Ramza J, Pucko W, Zegrocka-Stendel
O, et al. Cytostatic and cytotoxic activity of synthetic genistein glycosides against
human cancer cell lines. Cancer Lett 2004;203:5969.
[20] Blacon YC, Farias AS, Goelnitz U, Lopes SCP, Arrais-Silva WW, Carvalho BO, et al.
Hyperbaric oxygen prevents early death caused by experimental cerebral malaria.
PLoS One 2008;3:e3126.
[21] Zhou L, Ivanov II, Spolski R, Min R, Shenderov K, Egawa T, et al. IL-6 programs
T(H)-17 cell differentiation by promoting sequential engagement of the IL-21
and IL-23 pathways. Nat Immunol 2007;8:96774.
[22] Sakaguchi S, Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T. Regulatory T cells:
how do they suppress immune responses? Int Immunol 2009;21:110511.
[23] Chen W, Jin W, Hardegen N, Lei K, Li L, Marinos N. Conversion of peripheral CD4
+CD25 nave T cells to CD4+CD25+ regulatory T cells by TGF- induction of
transcription factor Foxp3. J Exp Med 2003;198:187586.
[24] Betelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, et al. Reciprocal develop-
mental pathways for the generation of pathogenic effector TH17 and regulatory T
cells. Nature 2006;441:2358.
[25] Wing K, Onishi Y, Prietro-Martin P, Yamaguchi T, Miyara M, Fehervari Z, et al.
CTLA-4 control over Foxp3
+
regulatory T cell function. Science 2008;322:2715.
[26] Takahashi T, Tagami T, Yamazaki S, Uede T, Shimizu J, Sakaguchi N, et al. Immuno-
logic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitu-
tively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med
2000;192:30310.
[27] Floris S, Blezer ELA, Schreibelt G, Dopp E, Van der Pol SMA, Schadee-Eestermans
IL. Bloodbrain barrier permeability and monocyte inltration in experimental
allergic encephalomyelitis: a quantitative MRI study. Brain 2004;127:61627.
[28] Uyttenhove C, Van Snick J. Development of an anti-IL-17A auto-vaccine that
prevents experimental auto-immune encephalomyelitis. Eur J Immunol
2006;36:286874.
[29] Komiyama Y, Nakae S, Matsuki T, Nambu A, Ishigame H, Kakuta S. IL-17 plays an
important role in the development of experimental autoimmune encephalomy-
elitis. J Immunol 2006;177:56673.
[30] Mangan PR, Harrington LE, Quinn DB, Helms WS, Bullard DC, Elson CO, et al.
Transforming growth factor- induces development of the Th17 lineage. Nature
2006;441:2314.
[31] Serada S, Fujimoto M, Mihara M, Koike N, Ohsugi Y, Nomura S, et al. IL-6 blockade
inhibits the induction of myelin antigen-specic Th17 cells and Th1 cells in
experimental autoimmune encephalomyelitis. PNAS 2008;105:90416.
[32] Renno T, Krakowski M, Piccirillo C, Lin JY, Owens T. TNF- expression by resident
microglia and inltrating leukocytes in the central nervous system of mice with
experimental allergic encephalomyelitis: regulation by Th1 cytokines. J Immunol
1995;154:94453.
[33] Juedes AE, Hjelmstrm P, Bergman CM, Neild AL, Ruddle NH. Kinetics and cellular
origin of cytokines in the central nervous system: insight into mechanisms of
myelin oligodendrocyte glycoprotein-induced experimental autoimmune en-
cephalomyelitis. J Immunol 2000;164:41926.
[34] Kohm AP, Carpentier PA, Anger HA, Miller SD. Cutting edge: CD4
+
CD25
+
regula-
tory T cells suppress antigen-specic autoreactive immune response and central
nervous system inammation during active experimental autoimmune encepha-
lomyelitis. J Immunol 2002;169:47126.
[35] Petro TM. Regulatory role of resveratrol on Th17 in autoimmune disease. Int
Immunopharmacol 2011;11:3108.
[36] Zhang R, Tian A, Zhang H, Zhou Z, Yu H, Chen L. Amelioration of experimental
autoimmune encephalomyelitis by -elemene treatment is associated with
Th17 and Treg cell balance. J Mol Neurosci 2011;44:3140.
[37] Cassan C, Piaggio E, Zappulla JP, Mars LT, Couturier N, Bucciarelli F. Pertussis toxin
reduces the number of splenic Foxp3
+
regulatory T cells. J Immunol 2006;177:
155260.
[38] Chen X, Winkler-Pickett RT, Carbonetti NH, Ortaldo JR, Oppenheim JJ, Howard
OMZ. Pertussis toxin as an adjuvant suppresses the number and function of
CD4
+
CD25
+
regulatory cells. Eur J Immunol 2006;36:67180.
[39] Zhang X, Koldizic DN, Izikson L, Reddy J, Nazareno RF, Sakaguchi S. IL-10 is
involved in the suppression of experimental autoimmune encephalomyelitis by
CD25
+
CD4
+
regulatory T cells. Int Immunol 2004;16:24956.
[40] Li R, Perez N, Karumuthil-Melethil S, Prabhakar BS, Holterman MJ, Chenthamarakshan
VasuC. Enhancedengagement of CTLA-4induces antigen-specic CD4
+
CD25
+
Foxp3
+
and CD4
+
CD25

TGF-1
+
adaptive regulatory T cells. J Immunol 2007;179:5191203.
[41] Hurwitz AA, Sullivan TJ, Sobel RA, Allison JP. Cytotoxic T lymphocyte antigen-4
(CTLA-4) limits the expansion of encephalitogenic T cells in experimental autoim-
mune encephalomyelitis (EAE)-resistant BALB/c mice. PNAS 2002;99:30137.
[42] Piao WH, Wong R, Bai XF, Huan GJ, Campagnolo DI, Dorr RT, et al. Therapeutic
effect of anthracene-based anticancer agent ethonade in an animal model of
multiple sclerosis. J Immunol 2007;179:741523.
[43] Kroenke MA, Carlson TJ, Andjelkovic AV, Segal BM. IL-12 and IL-23-modulated T
cells induce distinct types of EAE based on histology, CNS chemokine prole,
and response to cytokine inhibition. J Exp Med 2008;205:153541.
[44] Matsushita T, Yanaba K, Bouaziz JD, Fujimoto M, Tedder TF. Regulatory B cells
inhibit EAE initiation in mice while other B cells promote disease progression.
J Clin Invest 2008;118:342030.
[45] Kivisakk P, Mahad DJ, Callahan MK, Trebst C, Tucky B, Wei T, et al. Human
cerebrospinal uid central memory CD4+ T cells: evidence for trafcking
through choroid plexus and meninges via P-selectin. Proc Natl Acad Sci U S A
2003;100:838994.
[46] Wilson EH, Weninger W, Hunter CA. Trafcking of immune cells in the central
nervous system. J Clin Invest 2010;120:136879.
[47] Engelhardt B, Ransohoff RM. The ins and outs of T lymphocyte trafcking to the
CNS: anatomical sites and molecular mechanisms. Trends Immunol 2005;26:
48595.
470 S.B.R. Castro et al. / International Immunopharmacology 12 (2012) 465470