PHYTOTHERAPY RESEARCH

Phytother. Res. 29: 1707–1713 (2015)

Published online 14 July 2015 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.5414

Differential Inhibition of T Lymphocyte

Proliferation and Cytokine Synthesis by
[6]-Gingerol, [8]-Gingerol, and [10]-Gingerol

Megan Bernard,1 Suzanne J. Furlong,1 Melanie R. Power Coombs1 and David W. Hoskin1,2,3*
1
Department of Pathology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada
2
Department of Microbiology and Immunology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada
3
Department of Surgery, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada

[6]-Gingerol, [8]-gingerol, and [10]-gingerol are pungent components of fresh ginger, extracts of which inhibit various
components of the inflammatory response. Because little is known regarding the effect of gingerols with different
unbranched alkyl side chain lengths on the activation and effector function of T lymphocytes, we compared the effects
of [6]-gingerol, [8]-gingerol, and [10]-gingerol on murine T lymphocyte proliferation, expression of CD25 and CD69
activation markers, cytokine synthesis, and interleukin (IL)-2 receptor signaling. All three gingerols inhibited DNA
synthesis by T lymphocytes, as well as interferon-γ synthesis. In contrast, only [8]-gingerol and [10]-gingerol inhibited
CD25 and CD69 expression, and IL-2 synthesis. None of the gingerols affected IL-4 synthesis. Exogenous IL-2
enhanced T lymphocyte proliferation in the presence of [6]-gingerol but did not significantly increase T lymphocyte
proliferation in the presence of [8]-gingerol or [10]-gingerol. In line with this finding, [8]-gingerol and [10]-gingerol
impaired IL-2-induced proliferation of CTLL-2 cells, but constitutive CD25 expression was unaffected, indicating
inhibition of IL-2 receptor signaling. In general, [10]-gingerol and [8]-gingerol were more potent inhibitors of T
lymphocytes than [6]-gingerol. Suppression of T lymphocyte responses by gingerols suggests that these phytochemi-
cals may be beneficial in chronic inflammatory conditions associated with excessive or inappropriate T lymphocyte
activation. Copyright © 2015 John Wiley & Sons, Ltd.
Keywords: cytokine; DNA synthesis; inflammation; ginger; gingerol; T lymphocyte.

Abbreviations: Ab, antibody; DMSO, dimethylsulfoxide; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon;
IL, interleukin; LPS, lipopolysaccharide; NF-κB, nuclear factor κB; OVA, ovalbumin; PBS, phosphate buffered saline; TCR, T cell
3
receptor; [ H]TdR, tritiated thymidine

eosinophilia (Ahui et al., 2008). Ginger is also a rich

INTRODUCTION
Functional foods contain a plethora of bioactive compo-
nents, including phytochemicals that have potentially
beneficial effects on human health (Hasler et al., 2004).
Ginger (Zingiber officinale Roscoe), which is commonly
used as a spice and condiment, originated in South-East
Asia where the ginger rhizome has a long history of use
in traditional medicine (Butt and Sultan, 2011). Medici-
nal uses of ginger include the treatment of chronic
inflammatory conditions such as arthritis and rheuma-
tism, which is in line with increasing evidence that ginger
has broad antiinflammatory actions (Grzanna et al.,
2005). In this regard, ginger contains compounds that in-
hibit lipopolysaccharide (LPS)-induced prostaglandin E2
synthesis (Jolad et al., 2004), as well as the production of
inflammation-promoting cytokines and chemokines by
LPS-stimulated macrophages (Tripathi et al., 2008). In
addition, ginger extract suppresses ovalbumin (OVA)-in-
duced allergic airway inflammation in mice by interfering
with both Th1 and Th2 cytokine production, as well
as inhibiting IgE production and eotaxin-induced
* Correspondence to: David W. Hoskin, Department of Microbiology and
Immunology, Dalhousie University, 5850 College Street, PO Box 15000,
Halifax, Nova Scotia, Canada B3H 4R2.
E-mail: d.w.hoskin@dal.ca
source of antioxidants that prevent inflammation caused
by oxidative stress (Dugasani et al., 2010).
Analysis of fresh ginger rhizome by gas chromatography–
mass spectrometry reveals that gingerols, which are
pungent phenols with different unbranched alkyl side
chain lengths, are the predominant phytochemicals,
with [6]-gingerol being the most abundant, followed
by [10]-gingerol and [8]-gingerol (Jolad et al., 2004).
Shoagols, which are the dehydrated form of the corre-
sponding gingerols, are present in only small amounts
in fresh ginger rhizome. [6]-Gingerol is the most studied
of the gingerols in terms of antiinflammatory activities.
Topical application of [6]-gingerol to mouse skin
inhibits nuclear factor (NF)-κB-dependent expression of
cyclooxygenase-2 induced by phorbol 12-myristate 13-
acetate (Kim et al., 2004). Macrophage synthesis of LPS-
induced production of pro-inflammatory cytokines and
other inflammatory mediators such as prostaglandin E2
is also inhibited by [6]-gingerol (Tripathi et al., 2007;
Dugasani et al., 2010). Similar antiinflammatory effects
have been described for [8]-gingerol and [10]-gingerol
(Dugasani et al., 2010), including inhibition of NF-κB-
dependent pro-inflammatory cytokine expression by
microglial cells (Ho et al., 2013). In addition, [8]-gingerol
suppresses mitogen-driven proliferation of mouse
splenocytes and the OVA-specific humoral immune
response in OVA-immunized mice (Lu et al., 2011).

Copyright © 2015 John Wiley & Sons, Ltd.

1708 M. BERNARD ET AL.
However, little is known about the effect that gingerols
have on the activation and effector function of T lympho-
cytes, which are involved in the pathogenesis of a number
of autoimmune diseases and other chronic inflammatory
disorders (Cosmi et al., 2014). In this study, we investi-
gated the effect of [6]-gingerol, [8]-gingerol, and [10]-
gingerol on T lymphocyte activation and proliferation,
as well as cytokine production and signaling via the inter-
leukin (IL)-2 receptor.
MATERIALS AND METHODS
Animals. Female C57BL/6 mice aged 6–8 weeks were
purchased from Charles River Laboratory (Lasalle,
PQ) and housed at the Carleton Animal Care Facility
of Dalhousie University. Animal protocols were consis-
tent with the Canadian Council on Animal Care Guide-
lines and were approved by the Dalhousie University
Committee on Laboratory Animals. All mice used in
these experiments were 8–12 weeks old.
Reagents. [6]-Gingerol (purity > 95%) was purchased
from Dalton Pharma Services (Toronto, ON). [8]-Gingerol
and [10]-gingerol (purity > 98%) were from Chengdu
Biopurify Phytochemicals Ltd. (Chengdu, Sichuan, China).
Stock solutions of each gingerol were prepared at 300 mM
in dimethylsulfoxide (DMSO) and stored at 20 °C.
Phosphate buffered saline (PBS), LPS, bovine serum
albumin, and DMSO were purchased from Sigma-Aldrich
Canada (Oakville, ON). Functional grade hamster anti-
mouse T cell receptor (TCR) β, phycoerythrin-conjugated
anti-CD25, fluorescein isothiocyanate-conjugated anti-
CD69, phycoerythrin-conjugated rat IgG1, and fluorescein
isothiocyanate-conjugated Armenian hamster IgG anti-
bodies (Ab) were from eBioscience Inc. (San Diego,
CA). Recombinant mouse IL-2 was purchased from
PeproTech Inc. (Rocky Hill, NJ). Recombinant mouse
granulocyte-macrophage colony-stimulating factor
(GM-CSF) was from R&D Systems, Inc. (Minneapolis,
MN). Dynabeads® (anti-CD3 and anti-CD28 Ab-
coated microbeads) were from Invitrogen Canada Inc.
(Burlington, ON)
Cell line. IL-2-dependent mouse CTLL-2 CD8+ T lym-
phocytes (Gillis and Smith, 1977) were obtained from
American Tissue Culture Collection (Manassas, VA) and
maintained at 37 °C in a humidified incubator with 5%
CO2. CTLL-2 cells were cultured in RPMI 1640 medium
(Sigma-Aldrich Canada) supplemented with 5% heat-
inactivated fetal calf serum, 1% penicillin–streptomycin,
2 mM L-glutamine, and 5 mM HEPES (Invitrogen Canada
Inc.), hereafter referred to as complete RPMI 1640 me-
dium, and 50 U/mL recombinant mouse IL-2.
T lymphocyte isolation. Spleens were harvested from eu-
thanized C57BL/6 mice and homogenized in ice-cold PBS
to create a single cell suspension. Spleen cells were washed
in PBS, and erythrocytes were removed by hypo-osmotic
shock. CD3+ T lymphocytes were isolated using the Pan
T Cell Isolation MACS® kit from Miltenyi Biotech
Copyright © 2015 John Wiley & Sons, Ltd.
(Auburn, CA). After additional washes, T lymphocytes
were resuspended in complete RPMI 1640 medium. T
lymphocyte purity was at least 97%.
Dendritic cell generation. Bone marrow was flushed
from the tibia and femur of euthanized C57BL/6 mice
with ice-cold PBS and forced through an 18 gauge
needle to create a single cell suspension. Bone marrow
cells were washed and cultured for 7 days in complete
RPMI 1640 medium containing 20 ng/mL GM-CSF to
promote dendritic cell differentiation. Cells were then
washed and cultured for an additional 24 h in medium
containing 10 ng/mL GM-CSF and 1 μg/mL LPS in
order to generate mature dendritic cells.
T lymphocyte proliferation. T lymphocytes in complete
RPMI 1640 medium were plated in triplicate at 2.5 × 105
cells/well into 96-well round-bottom plates, treated as indi-
cated, and activated with Dynabeads® at a ratio of 1 bead
for every 2 T lymphocytes. In some experiments, syngeneic
dendritic cells (6.4 × 103 cells/well) and anti-TCRβ Ab
(20 μg/mL) were used instead of Dynabeads® to activate
T lymphocytes. Cultures were maintained for 48, 72, or
96 h at 37 °C in a humidified 5% CO2 atmosphere, and
pulsed with 0.2 μCi tritiated thymidine ([3H]TdR) from
MP Biomedicals (Irvine, CA) 6 h before the end of the
experiment. Cultures were then harvested onto fiberglass
filters with a Titertek® Cell Harvester (Skatron Instru-
ments, Sterling,VA) and [3H]TdR incorporation into
newly synthesized DNA was measured using a Beckman
LS6000IC liquid scintillation counter (Beckman Coulter
Inc., Mississauga, ON).
Cell viability. A Trypan Blue dye exclusion assay was
used to measure T lymphocyte and dendritic cell viabil-
ity after culture for 24 h in the absence or presence of
[6]-gingerol, [8]-gingerol, or [10]-gingerol. Cells were
washed and resuspended in medium and Trypan Blue
dye at a 1:1 ratio (v/v). A hemocytometer and light mi-
croscope were used to count live and dead cells; % live
cells = C / T × 100, where C is the number of live
unstained cells and T is the total number of cells.
Flow cytometry. Cells were washed with ice-cold PBS,
resuspended in ice-cold flow cytometry buffer consisting
of 0.2% sodium azide (w/v) and 1% bovine serum albu-
min in PBS, and labeled with the desired fluorochrome-
conjugated Ab or isotype-matched Ab (10 μg/mL) for
30 min at 4 °C in the dark. Cells were then washed twice
with flow cytometry buffer, fixed with 1% paraformal-
dehyde (v/v) in PBS, and analyzed with a FACSCalibur
flow cytometer (BD Biosciences). Data were processed
using FCS Express software (version 3.0; De Novo Soft-
ware, Thornhill, ON).
Cytokine measurement. T lymphocytes in complete
RPMI 1640 medium were plated in triplicate at
2.5 × 105 cells/well into 96-well round-bottom plates
and treated as indicated. After 24 h culture, cell-free
supernatants were collected and cytokine content was
Phytother. Res. 29: 1707–1713 (2015)
 T LYMPHOCYTE INHIBITION BY GINGEROLS 1709

determined by enzyme-linked immunosorbent assay. In-
terferon (IFN)-γ and IL-2 were measured using kits
from BD Biosciences (Mississauga, ON). IL-4 was mea-
sured with a kit from eBioscience Inc. (San Diego, CA).
Absorbance was read at 450 nm on an Expert 96 Micro-
plate Reader (Biochrom ASYS, Cambridge, UK) and
the concentration of cytokine in pg/mL was determined
using SOFTmax® Pro software (version 4.3; Molecular
Devices Corp. Sunnyvale, CA).
to provide costimulatory signals (Fig. 1B). Interestingly,
[10]-gingerol showed increased inhibitory activity under
these conditions. There was no significant effect of
gingerols at the concentrations used in this study on T
lymphocyte and dendritic cell viability after 24-h culture
(data not shown).
Effect of [6]-gingerol, [8]-gingerol, and [10]-gingerol on
CD25 and CD69 expression

Statistical analysis. Statistical analysis was performed
using a one-way analysis of variance (ANOVA) with a
Tukey–Kramer multiple parameters post-test using
GraphPad Prism analysis software (GraphPad Software
Inc., La Jolla, CA). Data were considered statistically
significant when p < 0.05.
RESULTS
Effect of [6]-gingerol, [8]-gingerol, and [10]-gingerol on
T lymphocyte proliferation
To determine the effect of gingerols with different chain
lengths on activation-induced T lymphocyte prolifera-
tion in the absence of antigen-presenting cells, mouse
T lymphocytes were stimulated with Dynabeads® in
the absence or presence of different concentrations of
[6]-gingerol, [8]-gingerol, or [10]-gingerol. DNA synthe-
sis after 48, 72, and 96 h of culture was measured by [3H]
TdR incorporation. As shown in Fig. 1A, there was a
time- and dose-dependent decrease in the proliferation
of gingerol-treated T lymphocytes, regardless of the
gingerol chain length. However, at a concentration of
100 μM, both [8]-gingerol and [10]-gingerol were more
inhibitory than [6]-gingerol. Similar results were ob-
tained when T lymphocytes were stimulated with anti-
TCRβ Ab in the presence of syngeneic dendritic cells
We next examined the effect of gingerols on the expres-
sion of CD25 and CD69, which are markers of T lym-
phocyte activation (Biselli et al., 1992). Fig. 2 shows
that the amount of CD25 expressed by Dynabead®-
stimulated T lymphocytes was decreased in the presence
of [6]-gingerol, [8]-gingerol, or [10]-gingerol, but was
only statistically significant for 50-μM [8]-gingerol and
[10]-gingerol. The percentage of CD25-expressing T
lymphocytes following Dynabead® stimulation was not
substantially affected by any of the gingerols. Similar re-
sults were obtained for CD69 expression, i.e. 50-μM [8]-
gingerol and [10]-gingerol caused a significant reduction
in CD69 expressed by Dynabead®-stimulated T lympho-
cytes while none of the gingerols affected the percentage
of CD69-expressing T lymphocytes.
Effect of [6]-gingerol, [8]-gingerol, and [10]-gingerol on
T lymphocyte cytokine synthesis
Significant inhibition of IFN-γ production by
Dynabead®-stimulated T lymphocytes was observed
with 25 or 50-μM [6]-gingerol, [8]-gingerol, or [10]-
gingerol (Fig. 3A). In contrast, only [8]-gingerol and
[10]-gingerol at 50 μM showed significant inhibition of
IL-2 production by Dynabead®-stimulated T lympho-
cytes (Fig. 3B). None of the gingerols were able to in-
hibit IL-4 synthesis by Dynabead®-stimulated T
lymphocytes (Fig. 3C).

Figure 1. Gingerols inhibit DNA synthesis by activated T lymphocytes. (A) T lymphocytes were cultured in the presence of the DMSO vehicle
or the indicated concentrations of [6]-, [8]-, or [10]-gingerol, and stimulated with Dynabeads® for 48, 72, and 96 h. (B) T lymphocytes were
co-cultured with syngeneic dendritic cells in the presence of the DMSO vehicle or the indicated concentrations of [6]-gingerol, [8]-gingerol, or
3
[10]-gingerol, and stimulated with anti-TCRβ Ab for 48, 72, and 96 h. (A, B) Cultures were pulsed with [ H]TdR for the last 6 h of culture and
harvested for liquid scintillation counting. Data shown are the average cpm ± SEM of at least three independent experiments; * p < 0.05
compared to the vehicle at the corresponding time point by one-way ANOVA with a Tukey–Kramer multiple parameters post-test.

Copyright © 2015 John Wiley & Sons, Ltd. Phytother. Res. 29: 1707–1713 (2015)
1710 M. BERNARD ET AL.

Figure 2. [8]-Gingerol and [10]-gingerol inhibit T lymphocyte expression of CD25 and CD69. T lymphocytes were cultured in the presence of
the DMSO vehicle or the indicated concentrations of [6]-gingerol, [8]-gingerol, or [10]-gingerol, and stimulated with Dynabeads® for 24 h. T
lymphocytes were then stained with phycoerythrin-conjugated anti-CD25 Ab, fluorescein isothiocyanate-conjugated anti-CD69 Ab, or the
appropriate isotype control Ab, fixed, and analyzed by flow cytometry. Data shown are the median fluorescence intensities (MFI) and the
% of (A) CD25-positive T cells or (B) CD69-positive T cells ± SEM from at least three independent experiments; * p < 0.05 when compared
to the vehicle at the corresponding time point by one-way ANOVA with a Tukey–Kramer multiple parameters post-test.

Effect of exogenous IL-2 on gingerol-mediated
inhibition of T lymphocyte proliferation
Because [8]-gingerol and [10]-gingerol inhibited IL-2
synthesis, we explored the effect of exogenous IL-2 on
the Dynabead®-stimulated proliferation of gingerol-
treated T lymphocytes. As shown in Fig. 4, the addition
of IL-2, at concentrations that are typically present in
activated T lymphocyte cultures, failed to substantially
affect the inhibition of Dynabead®-stimulated T lym-
phocyte proliferation caused by [8]-gingerol and [10]-
gingerol; however, IL-2 enhanced the proliferation of
T lymphocytes that were stimulated with Dynabeads®
in the presence of [6]-gingerol, in spite of the failure of
[6]-gingerol to decrease IL-2 production by these cells.
Effect of [6]-gingerol, [8]-gingerol, and [10]-gingerol on
IL-2 receptor signaling
To determine whether any of the gingerols affected IL-2
receptor signaling, we cultured IL-2-dependent CTLL-2
cells in the presence of 50 U/mL exogenous IL-2 with or
without increasing concentrations of [6]-gingerol, [8]-
gingerol, or [10]-gingerol. DNA synthesis was measured
after 48-h culture by [3H]TdR incorporation. There was
a statistically significant inhibition of IL-2-dependent
CTLL-2 cell proliferation in the presence of 100-μM
[8]-gingerol (vehicle—26 626 ± 3611 cpm versus [8]-
gingerol—4772 ± 1802 cpm, p < 0.005) and [10]-gingerol
(vehicle—27 123 ± 3582 cpm versus [10]-gingerol—5531
± 1396 cpm, p < 0.01). In contrast, 100-μM [6]-gingerol
did not affect IL-2-induced proliferation of CTLL-2 cells
Copyright © 2015 John Wiley & Sons, Ltd.
(vehicle—27 123 ± 3582 cpm versus [6]-gingerol—24 712
± 3448 cpm, p > 0.05). Inhibition of IL-2-dependent
CTLL-2 proliferation by [8]-gingerol and [10]-gingerol
was not because of a reduction in IL-2 receptor expres-
sion because constitutive expression of CD25 by CTLL-
2 cells was not decreased in the presence of 100-μM [8]-
gingerol or [10]-gingerol (data not shown).
DISCUSSION
Gingerols have potent antiinflammatory activities that may
be beneficial in individuals with chronic inflammatory
disorders (Tripathi et al., 2007; Dugasani et al., 2010). How-
ever, most studies to date have focused on the effect of
[6]-gingerol on pro-inflammatory functions of macrophages
with little attention being paid to the impact of gingerols on
T lymphocytes, which contribute to the pathogenesis of
many chronic inflammatory diseases (Cosmi et al., 2014).
This lack of information prompted us to compare the
effects of [6]-gingerol, [8]-gingerol, and [10]-gingerol on
the activation and effector function of T lymphocytes.
Non-cytotoxic concentrations of all three gingerols
inhibited mouse T lymphocyte proliferation induced by
Dynabeads®, although [8]-gingerol and [10]-gingerol
were generally more potent than [6]-gingerol. Gingerols
with different unbranched alkyl side chain lengths there-
fore have a direct inhibitory effect on T lymphocytes,
which is consistent with [8]-gingerol-mediated inhibition
of concanavalin A-driven mouse splenocyte prolifera-
tion (Lu et al., 2011). Interestingly, only [10]-gingerol
showed a further increase in inhibitory activity when T
Phytother. Res. 29: 1707–1713 (2015)
 T LYMPHOCYTE INHIBITION BY GINGEROLS 1711

Figure 3. Differential effect of gingerols on T lymphocyte cytokine synthesis. T lymphocytes were cultured in the presence of the DMSO ve-
hicle or the indicated concentrations of [6]-gingerol, [8]-gingerol, or [10]-gingerol, and stimulated with Dynabeads® for 24 h. Cell-free culture
supernatants were then collected for analysis by enzyme-linked immunosorbent assay. Data shown are the mean cytokine production of (A)
IFN-γ, (B) IL-2, and (C) IL-4, normalized to the medium control ± SEM from at least three independent experiments; * p < 0.05 when com-
pared to the vehicle at the corresponding time point by one-way ANOVA with a Tukey–Kramer multiple parameters post-test.

lymphocytes were stimulated with anti-TCRβ Ab in
the presence of syngeneic dendritic cells to provide
costimulatory signals, suggesting possible inhibition of
dendritic cell costimulatory function in addition to direct
inhibition of T lymphocytes.
We also observed substantial inhibition of activation-
induced T lymphocyte expression of CD25 and CD69
by [8]-gingerol and [10]-gingerol, but not [6]-gingerol,
which was in line with the stronger anti-proliferative ef-
fect of [8]-gingerol and [10]-gingerol. Because CD25 is
the α subunit of the high affinity interleukin-2 receptor
(Gaffen, 2001), it follows that T lymphocyte utilization
of IL-2 will be impaired in the presence of [8]-gingerol
or [10]-gingerol. Furthermore, both [8]-gingerol and
[10]-gingerol inhibited the IL-2 synthesis by T lympho-
cytes. Because IL-2 drives the clonal expansion of T
lymphocytes (Bayer et al., 2013), diminished utilization
of limited amounts of IL-2 likely accounts for much of
the inhibitory effect of [8]-gingerol and [10]-gingerol
on T lymphocyte proliferation. However, [8]-gingerol

Figure 4. Differential effect of exogenous IL-2 on gingerol-mediated inhibition of DNA synthesis by T lymphocytes. T lymphocytes were cul-
tured in the presence of the DMSO vehicle or 50-μM [6]-gingerol, [8]-gingerol, or [10]-gingerol without or with the indicated concentrations
3
of IL-2 and stimulated with Dynabeads® for 96 h. Cultures were pulsed with [ H]TdR for the last 6 h of culture and harvested for liquid scin-
tillation counting. Data shown are the average cpm ± SEM of at least three independent experiments; * p < 0.05 compared to the vehicle at
the corresponding time point by one-way ANOVA with a Tukey–Kramer multiple parameters post-test, n.s. denotes not significant.

Copyright © 2015 John Wiley & Sons, Ltd. Phytother. Res. 29: 1707–1713 (2015)
1712 M. BERNARD ET AL.
and [10]-gingerol also interfered with IL-2 receptor sig-
naling, which accounts for the failure of exogenous IL-2
to restore the proliferative response of gingerol-treated
T lymphocytes.
The profound inhibitory effect of [8]-gingerol and
[10]-gingerol on IL-2 synthesis and signaling suggests a
potential impact on regulatory T lymphocytes, which re-
quire IL-2 for their development and immunosuppressive
function (de la Rosa et al., 2004; Burchill et al., 2007). Such
an effect could at least partially counteract the inhibitory
effect of [8]-gingerol and [10]-gingerol on chronic inflam-
mation caused by inappropriate or excessive T lym-
phocyte activation and effector function. In contrast,
[6]-gingerol did not substantially affect IL-2 synthesis, in-
ducible CD25 expression, or IL-2 receptor signaling, and
the addition of exogenous IL-2 to T lymphocytes that
were stimulated in the presence of an inhibitory concen-
tration of [6]-gingerol resulted in increased DNA synthe-
sis. Although our data suggest that [6]-gingerol should not
interfere with regulatory T lymphocytes, tumor-bearing
mice treated with [6]-gingerol have a reduced number of
tumor-infiltrating regulatory T lymphocytes (Ju et al.,
2012). In contrast, numbers of tumor-infiltrating CD4+
and CD8+ T lymphocytes are dramatically increased fol-
lowing administration of [6]-gingerol; however, it is not
clear whether this effect was because of [6]-gingerol or a
[6]-gingerol metabolite (Nakazawa and Ohsawa, 2002).
T lymphocytes are an important source of cytokines
that drive immune responses; IFN-γ and IL-4 are repre-
sentative of Th1 and Th2 responses, respectively (Zhu
et al., 2010). Interestingly, [6]-gingerol, [8]-gingerol and
[10]-gingerol inhibited T lymphocyte synthesis of IFN-γ
whereas none of these gingerols affected IL-4 produc-
tion. These gingerols therefore have the potential to pref-
erentially inhibit Th1 responses that contribute to the
development of certain autoimmune diseases and other
chronic inflammatory disorders (Cosmi et al., 2014).
Our findings also suggest that reduced IFN-γ levels
reported in bronchoalveolar lavage fluids from
OVA-immunized and OVA-challenged mice (Ahui
REFERENCES
Ahui MLB, Champy P, Ramadan A, et al. 2008. Ginger prevents
Th2-mediated immune responses in a mouse model of airway
inflammation. Int Immunopharmacol 8: 1626–32.
Bayer AL, Pugliese A, Malek TR. 2013. The IL-2/IL-2R system:
from basic science to therapeutic applications to enhance
immune regulation. Immunol Res 57: 197–209.
Biselli R, Matricardi PM, D’Amelio R, et al. 1992. Multiparametric
flow cytometric analysis of the kinetics of surface molecule
expression after polyclonal activation of human peripheral
blood T lymphocytes. Scand J Immunol 35: 439–447.
Burchill MA, Yang J, Vogtenhuber C, et al. 2007. IL-2 receptor
β-dependent STAT5 activation is required for the development
+
of Foxp3 regulatory T cells. J Immunol 178: 280–290.
Butt MS, Sultan MT. 2011. Ginger and its health claims: molecular
aspects. Crit Rev Food Sci Nutr 51: 383–393.
Cosmi L, Maggi L, Santarlasci V, et al. 2014. T helper cells plastic-
ity in inflammation. Cytom Part A 85: 36–42.
De la Rosa M, Rutz S, Dorninger H, et al. 2004. Interleukin-2 is
+ +
essential for CD4 CD25 regulatory T cell function. Eur
J Immunol 34: 2480–2488.
Dugasani S, Pichika MR, Nadarajah VD, et al. 2010. Comparative anti-
oxidant and anti-inflammatory effects of [6]-gingerol, [8]-gingerol,
[10]-gingerol and [6]-shogaol. J Ethnopharmacol 127: 515–520.
Gaffen SL. 2001. Signaling domains of the interleukin 2 receptor.
Cytokine 14: 63–77.
Gillis S, Smith KA. 1977. In vitro generation of tumor-specific
cytotoxic lymphocytes. Secondary allogeneic mixed tumor
Copyright © 2015 John Wiley & Sons, Ltd.
et al., 2008) could be because of gingerol-mediated inhibi-
tion of IFN-γ production by OVA-reactive T lympho-
cytes. However, it is important to note that IFN-γ
production by Th1 cells is also important in protection
against intracellular pathogens such as viruses
(Romagnani, 1994), which our data suggest might be neg-
atively impacted by gingerols. In addition, the failure of
[6]-gingerol, [8]-gingerol, and [10]-gingerol to interfere
with IL-4 synthesis in our hands suggests that another
component of ginger extract is responsible for interfering
with the generation of Th2 cytokines during allergic air-
way inflammation (Ahui et al., 2008). Because the NF-κ
B family of transcription factors are essential for the acti-
vation of T lymphocytes (Ruland and Mak, 2003), and
[6]-gingerol, [8]-gingerol, and [10]-gingerol inhibit NF-κ
B in other cell types (Kim et al., 2004; Ho et al., 2013),
it seems likely that gingerol-mediated inhibition of NF-κ
B is at least in part responsible for the direct suppression
of T lymphocyte activation and effector function by
[6]-gingerol, [8]-gingerol, and [10]-gingerol.
In conclusion, the present study has established that
gingerols with different unbranched alkyl side chain
lengths directly inhibit mouse T lymphocyte prolifera-
tion and cytokine production, albeit with some notable
differences in potency and effects. Our findings suggest
that gingerols should be further investigated for their
possible use in the treatment of autoimmune diseases
and other chronic inflammatory disorders caused by
inappropriate or excessive T lymphocyte responses.
Acknowledgements
This work was supported by a grant to DH from the Natural Sciences
and Engineering Research Council of Canada.
Conflict of Interest
The authors have declared that there is no conflict of interest.
lymphocyte culture of normal murine spleen cells. J Exp Med
146: 468–482.
Grzanna R, Lindmark L, Frondoza CG. 2005. Ginger—an herbal
medicinal product with broad anti-inflammatory actions.
J Med Food 8: 125–132.
Hasler CM, Bloch AS, Thomson CA, et al. 2004. Position of the
American Dietetic Association: functional foods. J Am Diet
Assoc 104: 814–826.
Ho SC, Chang KS, Lin CC. 2013. Anti-neuroinflammatory capacity
of fresh ginger is attributed mainly to 10-gingerol. Food Chem
141: 3183–3191.
Jolad SD, Lantz RC, Solyom AM, et al. 2004. Fresh organically grown
ginger (Zingiber officinale): composition and effects on LPS-
induced PGE2 production. Phytochemistry 65: 1937–1954.
Ju SA, Park SM, Lee YS, et al. 2012. Administration of 6-gingerol
greatly enhances the number of tumor-infiltrating lympho-
cytes in murine tumors. Int J Cancer 130: 2618–2628.
Kim SO, Chun K-S, Kundu JK, et al. 2004. Inhibitory effects of [6]-
gingerol on PMA-induced COX-2 expression and activation of
NF-κB and p38 MAPK in mouse skin. Biofactors 21: 27–31.
Lu J, Guan S, Shen X, et al. 2011. Immunosuppressive activity of
8-gingerol on immune responses in mice. Molecules 16:
2636–2645.
Nakazawa T, Ohsawa K. 2002. Metabolism of [6]-gingerol in rats.
Life Sci 70: 2165–2175.
Romagnani S 1994. Lymphokine production by human T cells in
disease states. Annu Rev Immunol 12: 227–257.
Phytother. Res. 29: 1707–1713 (2015)
 T LYMPHOCYTE INHIBITION BY GINGEROLS 1713

Ruland J, Mak TW. 2003. Transducing signals from antigen
receptors to nuclear factor κB. Immunol Rev 193: 93–100.
Tripathi S, Maier KG, Bruch D, et al. 2007. Effect of 6-gingerol on
pro-inflammatory cytokine production and costimulatory mol-
ecule expression in murine peritoneal macrophages. J Surg
Res 138: 209–213.
Tripathi S, Bruch D, Kittur DS. 2008. Ginger extract inhibits LPS
induced macrophage activation and function. BMC Complement
Altern Med 8: 1.
Zhu J, Yamane H, Paul WE. 2010. Differentiation of effector CD4 T
cell populations. Annu Rev Immunol 28: 445–489.

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