A salicylate-based small molecule HS-Cm exhibits immunomodulatory effects and inhibits dipeptidyl peptidase-IV activity in human T cells

Article abstract of DOI:10.1016/j.ejphar.2014.01.049

Activated T cells are key players in chronic inflammatory diseases, including atherosclerosis. Salicylates, like aspirin, display not only anti-inflammatory, anti-thrombotic, anti-atherosclerotic activities, but also immunomodulatory effects in T cells at high dosages. Here, we aimed to identify potent immunomodulators for T cells through cell-based screening from a mini-library of 300 salicylate-based small molecules, and elucidate the mechanisms. Human peripheral blood T cells were isolated from buffy coat. Phorbol 12-myristate 13-acetate plus ionomycin (P/I) was used to stimulate T cells. Cytokine production was measured by enzyme-linked immunosorbent assays. T cell activation markers were determined by flow cytometry. The activation of transcription factors and kinases was analyzed by western blotting, electrophoretic mobility shift assay, or kinase assay. Through library screening, we identified a small molecule named HS-Cm [C₁₃H ₉ClFNO₂; N-(4-chloro-2-fluorophenyl)-2-hydroxybenzamide] that exhibited potent immunomodulatory effects on T cells with low cytotoxicity. In P/I-stimulated T cells, HS-Cm inhibited the production of interleukin-2, tumor necrosis factor-alpha, and interferon-gamma and suppressed the expression of surface activation markers CD25, CD69, and CD71, but not CD45RO. HS-Cm down-regulated DNA-binding activities of activator protein-1 and nuclear factor-kappa B, but not nuclear factor of activated T-cells, through inhibiting c-Jun N-terminal kinase/p38 and inhibitor of kappaB alpha (IκBα) kinase (IKK)/IκBα pathways, respectively. On the basis of structure-activity relationship, HS-Cm exerted considerable inhibition of dipeptidyl-peptidase IV/CD26 activity in T cells. Our results suggested that the small molecule HS-Cm exhibiting immunomodulatory effects on T cells may be useful for therapeutics in chronic inflammatory diseases, like atherosclerosis, diabetes and autoimmune arthritis.

Full text of DOI:10.1016/j.ejphar.2014.01.049

European Journal of Pharmacology 726 (2014) 124–132
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
A salicylate-based small molecule HS-Cm exhibits immunomodulatory
effects and inhibits dipeptidyl peptidase-IV activity in human T cells
a,b
c
d
b
b
Jun-Ting Liou , Hsu-Shan Huang , Meng-Lin Chiang , Chin-Sheng Lin , Shih-Ping Yang ,
e
a,f,
n
Ling-Jun Ho , Jenn-Haung Lai
Graduate Institute of Medical Science, National Defense Medical Center, Taipei, Taiwan, ROC
a
b
c
d
e
f
Division of Cardiology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC
School of Pharmacy, National Defense Medical Center, Taiwan, ROC
Graduate Institute of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan, ROC
Institute of Cellular and System Medicine, National Health Research Institute, Zhunan, Taiwan, ROC
Division of Allergy, Immunology and Rheumatology, Department of Internal Medicine, Chang Gung Memorial Hospital, Chang Gung University, No. 5,
Fusing St., Gueishan Township, Tao-Yuan County, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 August 2013
Received in revised form
Activated T cells are key players in chronic inflammatory diseases, including atherosclerosis. Salicylates,
like aspirin, display not only anti-inflammatory, anti-thrombotic, anti-atherosclerotic activities, but also
immunomodulatory effects in T cells at high dosages. Here, we aimed to identify potent immunomo-
dulators for T cells through cell-based screening from a mini-library of 300 salicylate-based small
molecules, and elucidate the mechanisms. Human peripheral blood T cells were isolated from buffy coat.
Phorbol 12-myristate 13-acetate plus ionomycin (P/I) was used to stimulate T cells. Cytokine production
was measured by enzyme-linked immunosorbent assays. T cell activation markers were determined by
flow cytometry. The activation of transcription factors and kinases was analyzed by western blotting,
electrophoretic mobility shift assay, or kinase assay. Through library screening, we identified a small
22 January 2014
Accepted 24 January 2014
Available online 31 January 2014
Keywords:
Salicylate
T lymphocyte
Atherosclerosis
Diabetes
9 2
molecule named HS-Cm [C13H ClFNO ; N-(4-chloro-2-fluorophenyl)-2-hydroxybenzamide] that exhib-
ited potent immunomodulatory effects on T cells with low cytotoxicity. In P/I-stimulated T cells, HS-Cm
inhibited the production of interleukin-2, tumor necrosis factor-alpha, and interferon-gamma and
suppressed the expression of surface activation markers CD25, CD69, and CD71, but not CD45RO. HS-
Cm down-regulated DNA-binding activities of activator protein-1 and nuclear factor-kappa B, but not
nuclear factor of activated T-cells, through inhibiting c-Jun N-terminal kinase/p38 and inhibitor of
Arthritis
Dipeptidyl-peptidase IV inhibitor
kappaB alpha (I
κ
B
α
) kinase (IKK)/I
κ
B
α
pathways, respectively. On the basis of structure-activity
relationship, HS-Cm exerted considerable inhibition of dipeptidyl-peptidase IV/CD26 activity in T cells.
Our results suggested that the small molecule HS-Cm exhibiting immunomodulatory effects on T cells
may be useful for therapeutics in chronic inflammatory diseases, like atherosclerosis, diabetes and
autoimmune arthritis.
&
2014 Elsevier B.V. All rights reserved.
1
. Introduction
effector cells, cytokines, signaling molecules or pathways (Tabas
and Glass, 2013).
Non-resolving inflammation, though not the primary cause,
Atherosclerosis is one of the best-known examples of chronic
inflammatory disease and remains a major cause of death in
humans worldwide (Hansson, 2005). T cells have been identified
in all stages of atherosclerotic lesions and are known to strongly
influence disease severity (Hansson et al., 2006). Through cytokine
production and interplay among different immune effector cells in
atherosclerotic plaque, activated T cells contribute to amplified
and sustained inflammation, lesion growth, and disease progres-
sion (Koltsova et al., 2012). Surprisingly, the activation of periph-
eral blood T cell is also demonstrated in patients with coronary
artery disease, in particular acute coronary syndrome (Caligiuri
et al., 1998; Methe et al., 2005). Thus, T cells may serve as targets
for the novel and unconventional anti-atherosclerotic therapy
contributes significantly to the pathogenesis of various chronic
inflammatory diseases (Nathan and Ding, 2010). The difficulty in
removing or identifying precise identity of the inflammatory
stimulus in these diseases provides a rationale for the develop-
ment of anti-inflammatory therapies, targeting specific immune
n
Corresponding author at: Division of Allergy, Immunology and Rheumatology,
Department of Internal Medicine, Chang Gung Memorial Hospital, Chang Gung
University, No. 5, Fusing St., Gueishan Township, Tao-Yuan County, Taiwan, ROC.
Tel.: þ886 2 8792 7135; fax: þ886 2 8792 7136.
E-mail address: laiandho@gmail.com (J.-H. Lai).
0
014-2999/$ - see front matter & 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejphar.2014.01.049

J.-T. Liou et al. / European Journal of Pharmacology 726 (2014) 124–132
125
(Goronzy and Weyand, 2006). Immunomodulatory agents that
2. Materials and methods
inhibit T cell activation and subsequent cytokine production may
provide anti-atherosclerotic effects. In addition to atherosclerosis,
activation of T cells has been implicated in the pathogenesis of
several chronic inflammatory disorders, such as diabetes, rheuma-
toid arthritis, chronic obstructive pulmonary disorder, and inflam-
matory bowel disease (Cosio et al., 2009; Hansson et al., 2006;
Monteleone et al., 2011; Sell et al., 2012; Smolen and Steiner,
2.1. Chemicals and reagents
The 300 synthetic small molecules were structurally similar
and consisted of core amino compounds (aniline) coupled with
carboxylic acids (salicylic acid) via peptide (amide) bonding and
finally synthesized with different modifications and conjugations.
The structure of HS-Cm is depicted in Fig. 1 and the synthesis
process is explained in Supplementary materials. All compounds
were dissolved in dimethyl sulfoxide (DMSO) at a stock concen-
tration of 100 mM. For each experiment, the stock solution was
further diluted in culture medium to the desired concentration
with a final DMSO concentration of 0.05% as indicated.
2
003). It is therefore reasonable to target T cells in the treatment of
these chronic inflammatory disorders.
Salicylate-based drugs, and in particular acetylsalicylic acid
(
best known as aspirin), are commonly employed for treating a
wide variety of inflammatory diseases, including atherosclerosis,
rheumatoid arthritis, and inflammatory bowel disease. Aspirin
is the most commonly used salicylate on account of its broad
therapeutic indications including prevention of myocardial infarc-
tion and ischemic stroke, treatment of pain, fever, and inflamma-
tory disorders (Awtry and Loscalzo, 2000). It is generally accepted
that the main therapeutic effect of aspirin comes from inhibition
of cyclooxygenase (COX) by acetylation and subsequent reduction
of prostanoids biosynthesis. However, it has been established that
the anti-inflammatory properties of aspirin involve additional
mechanisms independent of COX inhibition (Tegeder et al.,
2.2. Isolation of human peripheral blood T cells
Human T lymphocytes were isolated and purified by negative
selection from the buffy coat of whole blood obtained from the
Taipei Blood Bank (Taipei, Taiwan) according to our previous
report (Lai et al., 2001). After collection, T cells were maintained
in RPMI-1640 medium containing 10% fetal bovine serum (FBS)
and 1% penicillin/streptomycin at 37 1C with 5% CO and were used
2
for single experiments within 24 h.
2
001). Salicylate, the active metabolite of aspirin, may play more
important roles in the anti-atherosclerotic effects of aspirin
Jaichander et al., 2008). Moreover, aspirin has been shown to
(
reduce atherosclerosis by inhibiting fractalkine expression in
atherosclerotic plaques of murine models (Liu et al., 2010) provid-
ing more evidence for anti-atherosclerotic potential of salicylate.
The COX-independent pharmacological actions of aspirin and
salicylates demonstrated in different models are mediated by the
inhibition of several transcription factors, such as nuclear factor-
2
.3. Cell treatment and stimulation
Cells were pretreated with small molecules at various concen-
trations or 0.05% DMSO as a vehicle control for various time as
indicated. For cell activation, T cells were stimulated with 5 ng/mL
phorbol 12-myristate 13-acetate (PMA, Sigma, St. Louis, MO, USA)
kappa B (NF- B), activator protein-1 (AP-1), and nuclear factor of
κ
and 1
μM ionomycin (Sigma; PMAþionomycin: P/I). After stimu-
activated T-cells (NFAT), all essential signaling pathways for T-cell
proliferation, survival, and cytokine production (Aceves et al.,
lation, cell pellets or supernatants were collected at different time
points for analysis.
2
004; Kopp and Ghosh, 1994; Yin et al., 1998). Therefore, salicy-
lates with more potent immunomodulatory effects may provide
more therapeutic potential and benefit.
2.4. Measurement of cytokine production by enzyme-linked
immunosorbent assay (ELISA)
There is continuing interest in developing salicylate/salicylic
acid-based small molecules potential in tuning cellular response
for different therapeutic purposes (Kim et al., 2012). We previously
reported a novel small molecule HS-Cf with potential therapeutic
activity in the treatment of osteoarthritis (Liu et al., 2011). In the
current study, we used cell-based screening of the same mini-
library and identified another novel small molecule, designated
Human T cells were seeded in 24-well plate at a density of
6
1 ꢀ 10 cells/mL, followed by treatment with various compounds
and stimulation with P/I as indicated in the figure legends.
Concentrations of interleukin (IL)-2, interferon-gamma (INF-
γ
),
and tumor necrosis factor-alpha (TNF- ) in culture supernatants
α
HS-Cm [C13
H
9
ClFNO
2
;
N-(4-chloro-2-fluorophenyl)-2-hydroxy-
were measured by ELISA according to the manufacturer's instruc-
tions (R&D Systems, Minneapolis, MN, USA) and our previous
report (Yang et al., 2004). The IC50 value (concentration of
compound causing 50% inhibition of cytokine release) of each
compound was calculated by means of GraphPad Prism 4 (Graph-
Pad Software, San Diego, CA, USA).
benzamide], with unique immunomodulatory effects on human
T cells. Surprisingly, HS-Cm preserved considerable inhibitory
effect on the enzymatic activity of dipeptidyl peptidase IV
(
DPP4)/CD26. We also evaluated the molecular mechanisms of
HS-Cm on T-cell inhibition.
Fig. 1. Structure and chemical synthesis of HS-Cm. Chemical structures of HS-Cm [C13
H
9
ClFNO
2
; N-(4-chloro-2-fluorophenyl)-2-hydroxybenzamide], salicylic acid, 4-chloro-
0
4-fluoronaniline. DMF, dimethylformamide; HOBt, 1-hydroxybenzotriazole monohydrate; N-ethyl-N -(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).

126
J.-T. Liou et al. / European Journal of Pharmacology 726 (2014) 124–132
2
.5. Lactate dehydrogenase (LDH) cytotoxicity assays
encoding AP-1, NF-
USA) or NFAT (Promega, Madison, WI, USA) were used as DNA
probes. These DNA probes were radiolabeled with [ - P] ATP by
T4 kinase according to the manufacturer's instructions (Promega).
Competition assays with 100-fold molar excess of unlabeled AP-1
κ
B (Santa Cruz Biotechnology, Santa Cruz, CA,
6
32
Cytotoxicity of small molecule in human T cells (1 ꢀ 10 cells/mL in
γ
24-well plate) was estimated by measuring the release of LDH into the
culture medium. The LDH activity was quantified using a cytotoxicity
detection kit (LDH; Roche, Indianapolis, IN, USA). Maximum LDH
release was determined by lysing equal amounts of cells with 1%
Triton X-100 and spontaneous LDH release (low control) was deter-
mined by incubating cells with medium alone. The percent cytotoxi-
city was calculated as:ðexperimental releaseꢁ spontaneous releaseÞ=
ðmaximum release–spontaneous releaseÞ ꢀ 100:
or NF- B probes were performed to verify the binding specificity.
κ
Unlabeled nonspecific oligonucleotides (Santa Cruz Biotechnology)
were also included as a negative control.
2.8. Western blot analysis
Nuclear, cytoplasmic, and total lysate proteins were extracted
6
2.6. Flow cytometry analysis of cell-surface molecule expression
from treated cells (2 ꢀ 10 cells/mL in 10-cm culture dishes) and
quantified by Bradford Assay (Bio-Rad laboratories, Hercules, CA,
USA). Equal amounts of protein were subjected to western blot-
ting, as described previously (Lai et al., 2001; Liou et al., 2008).
Membranes were blotted with primary antibodies against c-jun
N-terminal kinase (JNK), extracellular signal-regulated kinase
After treatment as indicated in the figure legend, cells (5 ꢀ
5
10 cells/mL) were washed, collected, and stained with fluorescein
isothiocyanate (FITC)-conjugated anti-IL-2 receptor alpha (anti-IL-
or anti-CD25), anti-CD69, anti-CD71, anti-CD45RO, or FITC-
2
R
α
conjugated isotype-matched monoclonal antibodies (Pharmingen)
at 4 1C for 30 min. Cells were then washed with PBS and fixed with
0
molecules was determined using a flow cytometer (Becton Dick-
inson FACS Calibur) with CellQuest (Becton Dickinson) software.
The mean fluorescence intensity from 10,000 cells was acquired,
and the values were used to represent the expression levels of
surface molecules.
(ERK), p38, p65, p50, I
(Chemicon, Temecula, CA, USA), I
κB
α
(Santa Cruz Biotechnology),
β
-actin
κB
α
kinase (IKK; IKK or IKK
α
β),
.5% paraformaldehyde in PBS. The expression of cell-surface
phosphorylated JNK (p-JNK), p-ERK, p-p38, or p-c-Jun (Cell Signal-
ing Technology, Danvers, MA, USA) overnight at 4 1C. After
incubation with the appropriate secondary antibodies conjugated
to horseradish peroxidase (HRP; Chemicon), positive immunor-
eactivity was detected with the ECL substrate (Amersham
Bioscience) and exposed to BioMax Light Film (Kodak).
2.7. Electrophoretic mobility shift assay (EMSA)
2.9. Immunoprecipitation kinase assay
6
Human T cells (2 ꢀ 10 cells/mL) in 10-cm culture dishes and
A GST-I
fected with pGEX-4T-2-I
IKK (Ho et al., 2004). Rabbit polyclonal antibodies for IKK
sc-7218) and goat polyclonal antibodies for IKK (T20, sc-7330)
κ
B
α
-fusion protein purified from Escherichia coli trans-
was used as a substrate for IKK and
(H-744,
serum-starved (1% FBS) condition were treated as indicated in the
figure legend. EMSA of nuclear extracts was performed as detailed
in our previous report (Yang et al., 2003). Oligonucleotides
κBα
α
β
α
β
Fig. 2. HS-Cm inhibited cytokine production from stimulated T cells without significant cytotoxicity. (A) Human T cells were pretreated with indicated concentrations of HS-
Cm or vehicle (V; DMSO, 0.05%) in duplicate or triplicate for 16 h and were then stimulated with P/I for another 24 h. Cell culture supernatants were collected, and LDH
activities were measured. Maximum LDH (Max LDH) release was determined from equal amount of cells lysed with 1% Triton X-100, and spontaneous LDH release was
determined from untreated cells. The percent cytotoxicity was calculated as:ðexperimental releaseꢁspontaneous releaseÞ=ðmaximum release–spontaneous releaseÞ ꢀ 100:
Human T cells were pretreated with HS-Cm for 2 h (B, C) or 16 h (D). Following 24 h of P/I stimulation, IL-2, INF-γ, and TNF-α levels in the supernatants, indicating T cell
activation, were measured by ELISA. Data are expressed as the percent cytokine produced compared with that in the control group without pretreatment (set at 100%) and
n
are the average7SD of 3–5 independent experiments conducted in triplicate; Po0.05. The 50% inhibitory concentration (IC50) of HS-Cm (concentration of HS-Cm causing
50% inhibition of cytokine release) were estimated after 24 h of P/I stimulation and are presented on the tops of the panels.

J.-T. Liou et al. / European Journal of Pharmacology 726 (2014) 124–132
127
were purchased from Santa Cruz Biotechnology. The immunopre-
cipitation kinase assay was performed as detailed in our previous
report (Ho et al., 2004).
Dunnett's multiple comparison tests using GraphPad Prism 4
(GraphPad Software, San Diego, CA, USA). P values of less than
0.05 were considered statistically significant.
2
.10. DPP4 peptidase activity in human T cells
3
. Results
6
Human T cells (1 ꢀ 10 cells/mL) in 6-well plates were pre-
3.1. HS-Cm exhibited superior immunomodulatory effects in human
treated with 10
μM HS-Cm, 0.05% DMSO, or 50 M of the DPP4
μ
T cells
inhibitor diprotin A (DP-A) for 1 h. CD26/DPP4 peptidase activity
of treated cells was measured using the chromogenic substrate
glycyl-prolyl-para-nitroanilide (Gly-Pro-pNa; Sigma) according to
a previous report with modifications (Christopherson et al., 2003).
First, we performed cell-based screening of our salicylate-based
small-molecule library for potential anti-inflammatory com-
pounds. The inhibition of IL-2 release in activated human T cells
served as an indicator of the immunomodulatory effect of each
small molecule. As representative data shown in Fig. S1, one novel
small molecule HS-Cm exhibited the most potent inhibitory effects
of all the compounds on IL-2 production; this compound was
subjected to further analysis.
A well-known DPP4 inhibitor, DP-A (50 M dissolved in DMSO;
μ
final DMSO concentration of 0.4% in the culture medium; Sigma),
was used as a positive control to evaluate the inhibition of
peptidase activity. Data were obtained from two independent
experiments in triplicate, and representative results are shown
as the mean7standard error of the mean (S.E.M.) of all tests.
2.11. Statistical analysis
3.2. HS-Cm inhibited pro-inflammatory cytokine production without
significant cytotoxicity
All results were obtained from at least three independent
experiments and are expressed as the mean7SD unless other-
wise specified. Data were analyzed with one-way ANOVA plus
The results from LDH assay showed that HS-Cm was not toxic
to T cells at concentrations below 10 M (Fig. 2A). Further analysis
μ
Fig. 3. HS-Cm inhibited the expression of CD25, CD69, and CD71, but not CD45RO. Human T cells were pretreated with indicated concentrations of HS-Cm (1–10 μM) or
vehicle for 2 h (A, B) or 16 h (C, D) and were then stimulated with P/I for 24 h. Sorted cells were analyzed for expression of CD25, CD 69, CD71, or CD45RO by flow cytometry
to determine T-cell activation status. Mean fluorescence intensities (MFIs) are presented as means7SDs (n¼3 independent experiments, except for CD45RO where n¼2);
n
Po0.05 vs. stimulated T cells without HS-Cm pretreatment. Representative histograms are shown for staining with specific antibodies for untreated control (ctrl) and for
P/I-stimulated T cells with and without 10 μM HS-Cm pretreatment.

128
J.-T. Liou et al. / European Journal of Pharmacology 726 (2014) 124–132
Fig. 4. HS-Cm suppressed the DNA-binding activities of AP-1 and NF-κB, but not NFAT. Human T cells in serum-starved (1% FBS) condition were pretreated with indicated
concentrations of HS-Cm (0–10 μM) or vehicle for 2 h and were then stimulated with P/I for 2 h. The nuclear protein extracts were analyzed for DNA-binding activities of
AP-1 (A), NF-κB (B), and NFAT (C) by EMSA. As described in Section 2, competition assays (Cmpt) were also performed using unlabeled wild-type (Wt) and mutant (Mt)
oligonucleotide probes to identify the bands specific for individual transcription factors (competition assay data for NFAT were not shown). AP-1 and NF-κB bindings were
specific because they could be competed out with an unlabeled, identical oligonucleotide, but not with an unrelated, nonspecific oligonucleotide. The representative results
n
and statistics of densitometric intensities from at least three independent experiments were shown; Po0.05 vs. vehicle control.
Fig. 5. HS-Cm downregulated AP-1 activity through inhibition of JNK/p38 MAPK activation. Human T cells were pretreated with indicated concentrations of HS-Cm (0–
1
0 μM) or vehicle for 2 h and were then incubated with (þ) or without (ꢁ) P/I for 15 min (A) or 2 h (B). The expression of target proteins was analyzed by western blotting.
The prefixes p- and t- indicate phosphorylated (activated) protein and total protein, respectively. The representative results and statistics of densitometric intensities from at
least three independent experiments were shown; Po0.05 vs. vehicle control.
n

J.-T. Liou et al. / European Journal of Pharmacology 726 (2014) 124–132
129
by ELISA showed that not only IL-2 but also IFN-
productions were concentration-dependently inhibited by HS-Cm
γ
and TNF-
α
the activities of both AP-1 and NF-
concentration-dependently inhibited by HS-Cm (Fig. 4).
κ
B, but not NFAT, were
in P/I-stimulated T cells, with IC50 values of 6.2771.99, 5.417
2
.39, and 7.4471.12 M, respectively (Fig. 2B–D).
μ
3.5. HS-Cm suppressed the activation of JNK and p38, but not ERK
AP-1 activation is mainly controlled by the activation of
3.3. HS-Cm inhibited the expression of T-cell activation markers
mitogen-activated protein kinases (MAPKs) signaling pathways,
including JNK, ERK, and p38. Hence, MAPK activities in T cells were
evaluated by western blotting. As Fig. 5A showed, HS-Cm treat-
ment for 2 h inhibited the phosphorylation of JNK and p38, but not
ERK, in T cells stimulated by P/I for 15 min. The expression of
phosphorylated c-Jun, activated by phosphorylated JNK, was also
suppressed by HS-Cm (Fig. 5B). These results suggested that the
immunomodulatory effects of HS-Cm on T cells were partly
attributable to the suppression of the JNK/p38 MAPK/AP-1
pathway.
We next examined the surface expression of T-cell activation
marker influenced by HS-Cm using flow cytometry. As Fig. 3A–C
showed, the expression of CD25, CD69 and CD71, markedly
induced by P/I stimulation, was significantly reduced by HS-Cm
in a concentration-dependent manner. However, the slightly
induced CD45RO was not suppressed by HS-Cm (Fig. 3D).
3
.4. HS-Cm downregulated the DNA-binding activities of AP-1 and
NF- B, but not NFAT
κ
3.6. HS-Cm suppressed NF-
κB signaling through inhibition of IκBα
To study the molecular mechanisms of the immunomodulatory
effects of HS-Cm on T cells, we conducted EMSAs to examine the
kinase activity, I degradation, and p65/p50 nuclear translocation
κBα
DNA binding activities of AP-1, NF-
transcription factors for T cell activation. The results showed that
κ
B, and NFAT, three major
NF-
κ
B stays inactive and retains in the cytoplasm before
activation due to the binding by inhibitory protein
I
κ
Bα.
Fig. 6. HS-Cm inhibited the IKK-IκBα-NF-κB signaling pathway. Human T cells in serum starved (1% FBS) condition were pretreated with the indicated concentrations of
HS-Cm (0–10 μM) or vehicle for 2 h and were then stimulated with P/I for 2 h (A) or 15 min (B, C). In (A), cytoplasmic extracts (CEs) and nuclear extracts (NEs) from collected
cells were analyzed for the expression of NF-κB p50 and p65 subunits. In (B), total cell lysates were analyzed by western blotting for measurement of IκBα protein and β-actin
expression as an internal control. In (C), IKKα and IKKβ kinase activities were analyzed by immunoprecipitation kinase assays. Total cell lysates were immunoprecipitated
32
with anti-IKKα or anti-IKKβ antibodies. After sequential washing, GST-IκBα as a substrate and 10 mCi of [γ- P]ATP were added. After the kinase reaction, the reaction mixture
was analyzed for IKKα and IKKβ activity by autoradiography. For determination of IKKα and β-actin protein levels, immunoblotting (IB) assays were performed. The
n
representative results and statistics of densitometric intensities from at least three independent experiments were shown; Po0.05 vs. control.

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J.-T. Liou et al. / European Journal of Pharmacology 726 (2014) 124–132
The down-regulation of NF-
κ
B DNA-binding activity by HS-Cm in
A (Fig. 7C) are shown (Fig. 7D). The immunomodulatory effects
and mechanisms of HS-Cm in human T cells are summarized in
Fig. 8.
activated T cells suggested the inhibition of upstream signaling
molecules. Western blotting showed that HS-Cm reduced the
nuclear translocation and reversely increased cytoplasmic seques-
tration of both p50 and p65, two subunits of NF-
Further analysis of total cell lysates showed that I
levels increased in cells treated with HS-Cm, suggesting that I
degradation was inhibited (Fig. 6B). Immunoprecipitation kinase
assays were then employed to evaluate the activities of I
kinases (IKKs), which phosphorylate I and bring its degrada-
tion. As Fig. 6C showed, HC-Cm effectively blocked kinase activities
of both IKK and IKK
κ
B (Fig. 6A).
κBα
protein
4
. Discussion
κBα
T cells interact with other leukocytes via various cytokines,
κBα
participate in the regulation of atherogenic responses, and con-
tribute to atherosclerosis progression (Wigren et al., 2012). Ther-
apeutic approaches to inhibit pro-atherogenic cytokines produced
by activated T cells are clinically relevant in mitigating athero-
sclerosis (Ait-Oufella et al., 2011). To identify potent immunomo-
dulator for T cells, in the current study we screened 300 synthetic
salicylate-based small molecules using human peripheral blood T
cells as a primary target and identified a novel small molecule HS-
Cm exhibiting the most potent immunomodulatory effect but with
very low toxicity to human T cells. HS-Cm effectively suppressed T-
cell activation and inhibited the P/I-induced production of IL-2,
κBα
α
β
.
3.7. HS-Cm inhibited DPP4 activity on primary human T cells
In view of the correlation between DPP4 activity and T cell
activation (Ohnuma et al., 2008) and the structural similarities of
HS-Cm to DPP4 substrates and DPP4 inhibitors, we next analyzed
the effects of HS-Cm on DPP4 activity in human T cells. The well-
known DPP4 inhibitor diprotin A (50
DPP4 activity (Liu et al., 2009) was used as a positive control. The
results showed that DPP4 activity in T cells was effectively
μ
M) sufficiently suppressing
IFN-
γ
, and TNF- in T cells, potentially attenuating T cell-mediated
α
inflammatory processes, including atherosclerosis.
T cell activation leads to cell differentiation and expression of
various cell surface markers, clearly distinguishing activated and
naïve T cells (Shipkova and Wieland, 2012). The COX-2 specific
inhibition severely diminished early and late events of T cell activa-
tion, namely reduced CD25 and CD71 cell surface expression, IL-2,
suppressed by 10 M HS-Cm (Fig. 7A), suggesting the potential
μ
of HS-Cm as a DPP4 inhibitor analog and immunomodulatory
agent. For comparison, the chemical structures of HS-Cm (Fig. 7B),
DPP4 substrate Gly-Pro-pNa (Fig. 7B) and DPP4 inhibitor diprotin
Fig. 7. HS-Cm exhibited considerable inhibitory effect on DPP4 activity in human T cells. (A) Human T cells were pretreated with 10 μM HS-Cm, 0.05% DMSO, or 50 μM of the
DPP4 inhibitor diprotin A (DP-A) for 1 h. DPP4 activity of sorted cells was then analyzed in 96-well plates using the chromogenic substrate Gly-Pro-pNa. The pNA produced
by DPP4 cleavage was measured at 405 nm every 2 min according to a pNA standard curve. The representative results are shown as the mean7S.E.M. of all tests. Linear
regression curves were calculated, and the equations are presented. (B) Structure of HS-Cm and (C) the chemical structures of Gly-Pro p-nitroanilide (DPP4 substrate) and
diprotin A (DPP4 inhibitor).

J.-T. Liou et al. / European Journal of Pharmacology 726 (2014) 124–132
131
mechanisms of salicylate-based small molecules, in addition to anti-
inflammatory activities.
DPP4, identical to T-cell activation antigen CD26, is involved in
T-cell activation, costimulation, and immune regulation (Ohnuma
et al., 2008). DPP4 (CD26) inhibitors have become promising drugs
for treating inflammatory diseases due to their ability to inhibit T-cell
proliferation and cytokine production (Yazbeck et al., 2009). The
beneficial effects of DPP4 inhibitors on cardiovascular disease beyond
glucose control are attracting more attention lately (Zhong et al.,
2013). The inhibition of CD26 activity by HS-Cm implicated that
immunomodulatory effects of HC-Cm on human T cells probably
involves more complex mechanisms and may also provide cardio-
vascular benefits.
In conclusion, through cell-based compound screening of a
mini-library of 300 salicylate-based small molecules, HS-Cm was
identified as a candidate compound with potent immunomodula-
tory effects in activated T cells. HS-Cm exhibited low cytotoxicity,
but significantly reduced production of IL-2, IFN-
γ
, and TNF-
α
in
activated T cells. HS-Cm also suppressed the expression of cell
surface activation markers on T cells, including CD25, CD67, and
CD71, but not CD45RO. Further analysis showed that the DNA-
Fig. 8. The proposed mechanism for the immunomodulatory effect of HS-Cm in
human T cells. HS-Cm inhibited T cell activation and cytokine production through
suppressing phosphorylation of JNK and P38, and the kinase activities of both IKKα
and IKKβ, leading to downregulation of AP-1 and NF-κB DNA binding activities
respectively. HS-Cm also inhibited the enzymatic activity of DPP4/CD26.
binding activities of AP-1 and NF-
regulated by HS-Cm through suppression of the JNK/p38 MAPK
and IKK/I signaling pathways respectively (see Fig. S2 for an
illustration of the proposed mechanism). In addition, HS-Cm
markedly inhibited DPP4/CD26 activity in T cells. These results
demonstrated the potential of HS-Cm as an anti-inflammatory,
immunomodulatory, and anti-atherogenic agent targeting T cell
activation and as a DPP4 inhibitor analog.
κ
B, but not NFAT, were down-
κBα
TNF-
expression. COX-2 inhibition also inhibited the transcription from
NF- B and NFAT-dependent enhancers (Iniguez et al., 1999). Inter-
α
and IFN-
γ
production and cell proliferation, but not CD69
κ
estingly, in our study, P/I-induced up-regulations of CD25, CD69, and
CD71 on human T cells were all effectively suppressed by HS-Cm.
It implied the unique immunomodulatory mechanism of HC-Cm on
T cells different from COX-2 inhibition. Besides, the expression of
CD45RO, a surface marker of memory T cells, was not affected by HS-
Cm. The differential effects on these activation markers implied that
HS-Cm, though a significant inhibitor of early T-cell activation,
probably exerted minor effects on T-cell differentiation.
Acknowledgments
This work is supported in part by grants from the National
Science Council (NSC 101-2314-B-182A-103-MY3), Taipei, Taiwan,
ROC. The help from Dr. Han-Bin Huang, School of Public Health,
National Defense Medical Center, Taipei, Taiwan, ROC for statistical
analysis of DPP4 activity was highly appreciated.
NF- B and AP-1, not only essential signaling pathways for T-cell
κ
activation and differentiation, but also are critical regulators of
inflammatory responses, and both proteins are potential thera-
peutic targets for various inflammatory diseases, including ather-
osclerosis (Meijer et al., 2012; Pamukcu et al., 2011). The marked
suppression of AP-1 and NF-
κ
B DNA-binding activities in P/I-
Appendix A. Supporting information
stimulated human T cells by HS-Cm suggested the potent
anti-inflammatory properties of HS-Cm. AP-1 activity is mainly
regulated by MAPKs, including JNK, ERK, and p38 (Shaulian and
Karin, 2002). ERK regulates cell proliferation, differentiation, and
migration (Qi and Elion, 2005); however, JNK and p38 are
associated with cell responses to stress, apoptosis, and inflamma-
tion (Kaminska, 2005). Our data demonstrated that HS-Cm sup-
pressed JNK/p38 but not ERK activation in stimulated T cells,
providing additional evidence for the unique anti-inflammatory
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.ejphar.2014.01.
049.
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