The present study investigated the effects of 4-O-chloroacetyl-2,3-di-O-acetyl-6-O-levulinoyl-b-d-glucopyr- anosyl]-1-3-1-O-p-methoxyphenyl-2-deoxy-2-N-trichloroacetyl-4,6-O-ben-zylidene
Trang 1a lipopolysaccharide-induced Toll-like receptor-mediated signalling antagonist
Mani D Kalluri1,*, Praneel Datla2,*, Akshaya Bellary1,*, Khalander Basha1, Ashwani Sharma1, Anuradha Sharma1, Shiva Singh1, Shakti Upadhyay2and Vikram Rajagopal1
1 Drug Discovery and Development Group, Reliance Life Sciences Pvt Ltd, Navi Mumbai, India
2 EID Parry India Ltd, Chennai, India
Introduction
Humans have evolutionarily conserved immune
recep-tors, Toll-like receptors (TLRs), which line up as our
first defences against the invading foreign pathogens
TLRs are able to elicit an immune response due to their ability to recognize the pathogen-associated molecular patterns of microbial pathogens, such as
Keywords
inflammation; lipopolysaccharide;
monocytes; NF-jB; TLR signalling; tumour
necrosis factor-a
Correspondence
V Rajagopal, Drug Discovery and
Development Group, Reliance Life Sciences
Pvt Ltd, Dhirubhai Ambani Life Sciences
Center, Rabale, Navi Mumbai-400701, India
Fax: +91 22 67678099
Tel: +91 22 67678854
E-mail: vikram_rajagopal@relbio.com
*These authors contributed equally to this
work
(Received 9 December 2009, revised 16
January 2010, accepted 21 January
2010)
doi:10.1111/j.1742-4658.2010.07589.x
The regulation of cytokines and pro-inflammatory genes is an absolute essentiality to combat inflammatory diseases The present study investigated the effects of 4-O-chloroacetyl-2,3-di-O-acetyl-6-O-levulinoyl-b-d-glucopyr- anosyl]-(1-3)-1-O-(p-methoxyphenyl)-2-deoxy-2-N-trichloroacetyl-4,6-O-ben-zylidene-a-d-glucopyranoside (RSCL-0409), a novel small molecule Toll-like receptor (TLR) signalling antagonist, and its mechanism of action in human monocytic (THP-1) cells stimulated with lipopolysaccharide (LPS) In THP-1 and RAW264.7 cells, RSCL-0409 suppressed LPS-induced production of tumour necrosis factor-a (TNF-a) with a 50% inhibitory concentration of 10.6 lm and mRNA expression of ICAM-1, Cox-2 and interleukin-8 with no evidence of cytotoxicity RSCL-0409 also suppressed TNF-a production from LPS-stimulated human peripheral blood mononuclear cells Similar results were obtained in vivo in a murine model of LPS-induced inflam-mation, where pretreatment with RSCL-0409 resulted in significant inhibi-tion of TNF-a It is also noteworthy that RSCL-0409 suppressed the cytokine production induced by TLR2 and -4 ligands and not for any other TLR ligands RSCL-0409 significantly inhibited p65 nuclear translocation induced by LPS In conclusion, RSCL-0409, a novel small molecule, is the first of its kind in the category of carbohydrate-derived TLR signalling anta-gonists and could definitely be a promising therapeutic agent for inflam-matory diseases whose pathogenesis involves TLR2- or TLR4-mediated signalling processes
Abbreviations
IL, interleukin; IRAK, interleukin-1 receptor-associated kinase; LBP, lipopolysaccharide-binding protein; LPS, lipopolysaccharide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; NF-jB, nuclear factor-jB; NO, nitric oxide; PBMC, peripheral blood mononuclear cell; RSCL-0409, [4-O-chloroacetyl-2,3-di-O-acetyl-6-O-levulinoyl-b- D -glucopyranosyl]-(1-3)-1-O-(p-methoxyphenyl)-2-deoxy-2-N-trichloroacetyl-4,6-O-benzylidene-a- D -glucopyranoside; SEAP, secreted embryonic alkaline phosphatase; TIRAP, Toll receptor IL-1R domain-containing adapter protein; TLR, Toll-like receptor; TNF-a, tumour necrosis factor-a; TRAF, TNFR-associated factor 6.
Trang 2bacteria and viruses, triggering inflammatory and
antiviral responses that destroy the invading
patho-gens So far, 11 TLRs have been described and for
most of them, except TLR10, natural ligands have
been identified These include various proteins,
lipo-peptides, lipoteichoic acid, lipopolysaccharides (LPS)
and oligonucleotides (double-stranded RNA,
single-stranded RNA and DNA) [1] One of the prominent
signalling mechanisms activated through TLRs is via
LPS LPS, the predominant structural component of
the outer membrane of Gram-negative bacteria,
acti-vates monocytes and macrophages, leading to the
pro-duction of cytokines, such as tumour necrosis factor-a
(TNF-a), interleukin-1 (IL-1) and IL-6, which in turn
serve as endogenous inflammatory mediators
Although TLR4 is primarily implicated as the TLR
involved in LPS signalling, there are reports showing
the involvement of TLR2 as the primary signal
tran-ducing molecule in Gram-negative organisms, such as
Porphyromonas gingivalis [2], which also have LPS as
the cell component Furthermore, studies have also
shown TLR1 functioning as a coreceptor for TLR2
and its coexpression in transfected cells augmented
the TLR4-independent response to Escherichia coli
LPS [3]
The cells become activated when the lipid A moiety
of LPS attaches to the LPS-binding protein (LBP); this
LPS⁄ LBP complex binds to CD14, which is then
trans-ferred to the TLR4–MD-2 complex [4,5] Activation of
mitogen-activated protein kinases and nuclear
factor-jB (NF-factor-jB) together with the release of inflammatory
mediators [6] are the result of the signal transduction
The LPS signalling cascade involves a lot of adapter
molecules, such as MyD88 [7] and Toll receptor IL-1R
domain-containing adapter protein (TIRAP)⁄ MyD88
adapter-like (Mal) These further recruit kinases,
IL-1R-associated kinase (IRAK)-1 and⁄ or IRAK-2 [8],
which in turn activate TNF receptor-associated factor
6-dependent signalling cascades, culminating in NF-jB
activation [9] and the release of various inflammatory
mediators Prominent among them is TNF-a capable
of exerting host-damaging effects seen in conditions
such as sepsis, fever syndromes, cachexia and in
auto-immune diseases such as rheumatic arthritis and
inflammatory bowel disease [10,11]
Given the wide implications of TLR signalling, its
regulation can alleviate the effects of pro-inflammatory
mediators [12] It is well documented that most known
TLR ligands contain carbohydrate moieties However,
the potential role of pure carbohydrates or its
analogues as ligands for TLRs has unlimited scope of
further investigation Documented reports show low
molecular mass hyaluronic acid oligosaccharides
pro-duced during inflammation exhibiting the ability to induce maturation of dendritic cells through TLR4 [13,14] Our goal was to use the available data to design novel selective carbohydrate-based, especially disaccharide-like molecules, that could serve either as agonists or antagonists of TLR signalling pathways Working in that direction, we discovered a novel disaccharide derivative, [4-O-chloroacetyl-2,3-di-O-acetyl- 6-O-levulinoyl-b-d-glucopyranosyl]-(1-3)-1-O-(p-meth- oxyphenyl)-2-deoxy-2-N-trichloroacetyl-4,6-O-benzy-lidene-a-d-glucopyranoside (RSCL-0409), from the vast library of analogues synthesized, which selec-tively inhibits the TLR-mediated production of TNF-a and also mRNA expression of various pro-inflammatory genes that lead to NF-jB activation The chemical structure of RSCL-0409 is shown in Fig 1A We have also delineated the mechanism responsible for the inhibitory effect
Results
RSCL-0409 inhibits the production of inflammatory cytokine TNF-a from LPS-stimulated THP-1, peripheral blood mononuclear cells (PBMCs) and RAW264.7 cells Human monocytic cells, THP-1, were stimulated with
250 ngÆmL)1LPS in the presence and absence of vari-ous concentrations of RSCL-0409 (1–100 lm) TNF-a
in the supernatants was measured by Duo-Set ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions RSCL-0409 inhibited the production of this pro-inflammatory mediator in a concentration-dependent manner (Fig 2A); having a 50% inhibitory concentration value of 10.6 lm (calcu-lated using biodatafit software) We observed similar inhibition in both RAW264.7 cells and PBMCs stimu-lated with LPS (Fig 2B, C) In addition, we checked its ability to inhibit TNF-a when stimulated with higher concentrations of LPS As shown in Fig 2E, RSCL-0409 at 10 lm showed an inhibition of TNF-a secretion even with 1000 ngÆmL)1LPS stimulation and
a near complete inhibition of TNF-a secretion observed at 100 lm, making it a potential candidate for use in clinical conditions such as septic shock where LPS concentrations are known to be very high Cell viability as estimated by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method (Fig 2D) did not show any cytotoxicity These results clearly indicate that RSCL-0409 exerts an inhib-itory effect on the production of the pro-inflammatory mediator TNF-a from both mouse and human monocytes and macrophages stimulated with LPS
Trang 3Inhibitory Effect on TLR1⁄ 2, -4 ligand-induced
TNF-a
The activation of TLRs by specific ligands leads to the
release of many inflammatory cytokines Studies have
shown that THP-1 cells are known to express all the
TLRs Therefore, we checked the effect of various
TLR ligands on THP-1 monocytes in the presence and
absence of RSCL-0409 (50 lm) and their ability to
release TNF-a As shown in Fig 3A, we detected
TNF-a secretion from cells stimulated with TLR1⁄ 2,
TLR4 and TLR6 No detectable TNF-a was observed
following stimulations with other ligands In cells pre-treated with RSCL-0409, we observed inhibited TNF-a production from THP-1 cells stimulated with TLR1⁄ 2 and TLR4 We confirmed this selective inhibitory effect of RSCL-0409 on TLR-1⁄ 2- and TLR4-mediated TNF-a production in PBMCs (Fig 3B) and a similar observation was also made in RAW264.7 cells (unpub-lished data) These results suggest that RSCL-0409 inhibits TNF-a production mediated by TLR1⁄ 2 and TLR4
To confirm the above observation, we checked for the ability of RSCL-0409 to inhibit nitric oxide (NO)
Chemical structure – RSCL-0409 A
B
Fig 1 (A) Structure of RSCL-0409 (B) HPLC profile of RSCL-0409 RSCL-0409 was synthesized as mentioned in Materials and methods The purified compound was subjected to HPLC to determine its purity Peak 4 with a retention time 3.6 min shows 98.6% purity.
Trang 4A B
C
E
D
Control
Control
LPS LPS
RSCL-0409 (1, 10, 50, 100 µ
M )
RSCL-0409 (1 µ
M ) + LPS
RSCL-0409 (10 µ
M ) + LPS
RSCL-0409 (50 µ
M ) + LPS
RSCL-0409 (100 µ
M ) + LPS
Control
Cells LPS
LPS
RSCL-0409 (1, 10, 50, 100 µ
M )
RSCL-0409 (1 µ
M ) + LPS
RSCL-0409 (10 µ
M ) + LPS
1 µ
M RSCL-0409 + LPS
10 µ
M RSCL-0409 + LPS
50 µ
M RSCL-0409 + LPS
100 µ
M RSCL-0409 + LPS
RSCL-0409 (50 µ
M ) + LPS
RSCL-0409 (100 µ
M ) + LPS
RSCL-0409
RSCL-0409 (1 µ
M )
RSCL-0409 (10 µ
M )
RSCL-0409 (50 µ
M )
RSCL-0409 (100 µ
M ) RSCL-0409 + LPS
250 ng·mL –1
500 ng·mL –1
1000 ng·mL –1
***
3000
2500
2000
1500
1000
500
0
NS
***
***
***
NS
*
***
***
2000
1500
1000
500
0
2500
3000
2000
1500
1000
500
0
1400
1600
1200
1000
800
600
400
200
0
125
100
75
50
25
0
Fig 2 RSCL-0409 suppression of LPS-induced TNF-a in a dose-dependent manner and in different cell lines Serum-starved human mono-cytic cells (THP-1; B), human PBMCs (B) and mouse macrophage cells (RAW264.7; C) were pretreated with the indicated concentrations of RSCL-0409 and then stimulated with LPS for 24 h Culture supernatants were collected and assayed for TNF-a by ELISA Data are expressed as means + standard error of three independent experiments *P < 0.05, ***P < 0.001; LPS treated vs RSCL-0409 treated, NS, not significant (D) Serum-starved THP-1 cells (2 · 10 5
cellsÆwell)1) were pretreated with RSCL-0409 in increasing concentrations (1, 10, 50 and 100 l M ) 1 h before LPS stimulation The viability of the cells was checked using MTT after 24 h incubation The data represented are after the values have been normalized to the control Error bars represent the standard error for three separate experiments (E) Serum-starved THP-1 cells were stimulated with increasing concentrations of LPS (250–1000 ngÆmL)1) with and without pretreatment with RSCL-0409 (1–100 l M ) for 24 h The supernatant was then assayed for the ability of the cells to release TNF-a by ELISA The data are a rep-resentation of two independent experiments.
Trang 5induced by TLR ligands in RAW264.7 cells
RSCL-0409 selectively inhibited TLR1⁄ 2- and LPS-induced
NO production (Fig 3C) RSCL-0409 had no
signifi-cant effect on NO production induced by TLR3,
TLR6, TLR7⁄ 8 or TLR9 ligands As shown in
Fig 3D, RSCL-0409 inhibited LPS-stimulated NO
production in a dose-dependent manner
Inhibitory effect on mRNA expression in THP-1
cells
To determine whether the suppressive effect of
RSCL-0409 on cytokine production occurs at the mRNA
expression level, we used quantitative real-time PCR to
examine TNF-a and IL-6 mRNA expressions in
THP-1 cells stimulated with LPS TNF-a (Fig 4A) and IL-6
(Fig 4B) mRNA expression was detected 1 h after
LPS stimulation The expression levels were similar to
cell control in RSCL-0409 (50 lm) pretreated cells On
the other hand, TNF-a and IL-6 mRNA showed
13- and six-fold increased expression after LPS stimu-lation, respectively Furthermore, we wanted to deter-mine whether this inhibitory effect on mRNA expression was seen on other pro-inflammatory genes, such as ICAM-1, Cox-2 and IL-8 We saw suppression
of their mRNA expression levels at a concentration of
50 lm RSCL-0409 (Fig 4C) It is also noteworthy that treatment of cells with RSCL-0409 did not show any effect in any of the genes at the mRNA level (Fig 4C, lane 2)
RSCL-0409 blocks nuclear translocation of NF-jB and activation of NF-jB transcription factor LPS, together with a range of inflammatory stimuli, activates and induces nuclear translocation of NF-jB NF-jB⁄ IjB complexes are present in the cytoplasm under unstimulated conditions Following stimulation with LPS, we see phosphorylation and subsequent deg-radation of IjB, allowing the free NF-jB to
C
D
Contr ol LPS
RSCL-0409 (1.25 µ
M ) + LPS
RSCL-0409 (2.5 µ
M ) + LPS
RSCL-0409 (5 µ
M ) + LPS
RSCL-0409 (10 µ
M ) + LPS
***
**
NS
**
**
NS
*
***
NS
NS
NS NS
*
***
***
***
0
500
1000
1500
2000
2500
3000
Effect of RSCL-0409 on various TLR ligands in THP-1 cells
Ligands alone
RSCL-0409
0 200 400 600 800 1000 1200 1400
Effect of RSCL-0409 on various TLR ligands in PBMCs
0
5
10
15
20
25
30
35
Contr
ol
Pam3CSK4.3HCl
Po ly(I:C)
Malp-2
CpG ODN 2395
0.0 4.0 8.0 12.0 16.0 20.0 24.0
Ligands alone RSCL-0409
Ligands alone RSCL-0409
Fig 3 RSCL-0409 inhibits TLR2, TLR4-induced TNF-a secretion in THP-1 monocytes and PBMCs Serum-starved THP-1 cells (A) and PBMCs (B), 2 · 10 5 cellsÆwell)1, were pretreated with RSCL-0409 (50 l M ) 1 h before TLR ligand treatment The pretreated cells were stimu-lated with various TLR ligands at different concentrations (TLR1 ⁄ 2 75 ngÆmL)1, TLR3 75 lgÆmL)1, TLR4 750 ngÆmL)1, TLR5 75 ngÆmL)1, TLR6 75 ngÆmL)1, TLR7 ⁄ 8 7.5 lgÆmL)1and TLR9 7.5 lgÆmL)1) for 24 h as per manufacturer’s instructions The culture supernatant was then assayed for TNF-a secretion Similar treatments were carried out on RAW 264.7 cells (C, D) We have used imidazoquinolines as TLR-7 ⁄ 8 for stimulation Cells untreated with RSCL-0409 served as controls Data are expressed as mean + standard error of two independent exper-iments *P value < 0.05, **P value < 0.01, ***P value < 0.001; ligand-treated cells vs RSCL-0409-treated cells, NS, not significant.
Trang 6cate into the nucleus to activate genes with
NF-jB-binding regions Therefore, we checked whether
RSCL-0409 blocked any signals responsible for NF-jB
signalling leading to nuclear translocation RSCL-0409 prevented IjB-a degradation and phosphorylation of the p65 subunit (data not shown), resulting in the
A
C
B
Cells +
+ LPS
Cells + LPS
Cell control Cells +
RSCL-0409 (50 µ M )
Cells +
RSCL-0409 (50 µ M ) + LPS Cells + LPS
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
6.0
5.0 4.0 3.0 2.0 1.0 0.0
7.0
9.0 IL-6
NS
***
8.0
ICAM-1
Control RSCL-0409 LPS RSCL-0409 + LPS
Fold change
Cox-2
Control RSCL-0409 LPS RSCL-0409 + LPS
Fold change
IL- 8
Control RSCL-0409 LPS RSCL-0409 + LPS
Fold change
ICAM-1
IL-8
Cox-2
β-actin
+
313 bp
221 bp
282 bp
278 bp
Fig 4 Inhibitory effect of RSCL-0409 on mRNA expression in THP-1 cells (A, B) Total RNA was isolated from THP-1 cells 1 h after expo-sure to LPS (250 ngÆmL)1) with or without 50 l M RSCL-0409 The cDNA was used for real-time PCR with primers specific for human TNF-a, IL-6 and for the housekeeping gene b-actin The fold change of TNF-a and IL-6 mRNA in treated cells over control was obtained after correc-tion for the amount of b-actin Error bars represent the standard error for three separate experiments ***P value < 0.001; LPS-treated cells
vs RSCL-0409-treated cells, NS, not significant (C) cDNA from an experiment similar to (A) was used to amplify inflammatory genes (ICAM-1, Cox-2 and IL-8) using specific primers (see Materials and methods) with b-actin serving as the internal control The data are representative of three independent experiments.
Trang 7inhibition of downstream signalling Further
down-stream, we saw partial translocation of NF-jB from
the cytoplasm into the nucleus within 60 min, which
was blocked when cells were pretreated with
RSCL-0409, as shown in Fig 5A
We confirmed the suppressive effect of RSCL-0409
on the NF-jB signalling pathway using an NF-jB
reporter assay In THP-1 blue CD14 cells containing
stably transfected NF-jB reporter plasmids expressing
the secreted embryonic alkaline phosphatase (SEAP)
gene on stimulation with a TLR4 ligand, LPS released
large amounts of SEAP into culture medium, which
was blocked in a dose-dependent manner by
pretreat-ment of cells with RSCL-0409 (Fig 5B) These
observations further confirm that RSCL-0409 inhibits
LPS-induced TLR-mediated activation of NF-jB
transcription factor
Effect of RSCL-0409 on LPS-induced TNF-a
production in vivo
To follow-up on our in vitro observations, we tested
the effect of RSCL-0409 pretreatment in a murine
model of LPS-induced inflammation Balb⁄ C was
trea-ted with RSCL-0409, 30 min before LPS injection
Blood samples were taken 1 h after LPS injection for
TNF-a analysis As shown in Fig 6, we saw 53 and
64% inhibition of LPS-induced TNF-a production at
1 h at 10 and 20 mgÆkg)1 injected RSCL-0409,
respec-tively
Discussion
The release of multiple pro-inflammatory
cytochemo-kines is a signature feature of the pathophysiology of
inflammation induced by LPS These processes, which
involve a number of signalling molecules, bring along
with them their share of complexities Thus, a good
anti-inflammatory agent targeting such a complex
sig-nalling mechanism needs to modulate this
pro-inflam-matory process, preferably at an early stage of
signalling Our research focuses on identifying such
novel molecules and taking them forward as novel
anti-inflammatory drugs
In our current study, we synthesized a library of
novel small carbohydrate-derived analogues and
identi-fied a novel gluco-disaccharide derivative, RSCL-0409,
through our screening process that exhibits strong
anti-inflammatory properties both in in vitro and
in vivo It inhibits LPS-induced signalling in cultured
monocytes and reduces NF-jB-dependent expression
of pro-inflammatory genes, ICAM-1, Cox-2 and IL-8,
through a TLR-mediated process It is of significance
that RSCL-0409 exerts similar inhibitory effects in both human monocytes and mouse macrophages, indi-cating that its potency is spread across different species
With TLRs gaining prominence as drug targets, numerous agonists are currently being developed [15]
0.0 20.0 40.0 60.0 80.0 100.0 120
NS NS NS
NS
**
***
.0
Control LPS-30 LPS-60 LPS-120 RSCL-0409+LPS-15 RSCL-0409+LPS-30 RSCL-0409+LPS-60
Fold change
Lanes
LPS RSCL-0409
1 2 3 4 5 6 7
0 30 60 120 30 60 120 +
– – – – – + + + + +
+ + +
p65NF-kB A
B
Tubulin
LPS Cell control
Cells + RSCL-0409 (1 µ
M )
Cells + RSCL-0409 (10 µ
M )
Cells + RSCL-0409 (50 µ
M )
Cells + RSCL-0409 (1 µ
Cells + RSCL-0409 (10 µ
Cells + RSCL-0409 (50 µ
Time (min)
Fig 5 RSCL-0409 blocks nuclear translocation of NF-jB and NF-jB activation in reporter assays (A) Serum-starved THP-1 cells were treated with 50 l M RSCL-0409 The nuclear fractions were obtained from LPS-stimulated THP-1 cells at the indicated times and processed for immunoblots as mentioned earlier using a NF-jB-specific antibody The blot was stripped and reprobed with
an anti-tubulin Ig to ensure equal loading All the results shown are representative of three separate experiments (B) THP-1 CD14 blue cells (Invivogen) transfected with a SEAP reporter construct, in which the reporter expression was regulated by the NF-jB pro-moter, were stimulated with LPS (250 ngÆmL)1) with or without RSCL-0409 for 24 h The reporter activity was determined using the Quanti Blue kit The data are plotted as the relative change in reporter activity The data shown are the average from three inde-pendent experiments performed in duplicate NS, not significant
**P value < 0.01, ***P value < 0.001; cells vs RSCL-0409 treated; LPS treated vs RSCL-0409 followed by treated.
Trang 8The development of antagonists has gained impetus
following concerns that agonists have not been
effec-tive in specific targeting Currently, TAK-242 [16] and
E-5564 [17], apart from the few reported lipid A
ana-logues as antagonists [18–20], are in the last phase of
clinical developmental Furthermore, reports of
LPS-like molecules extracted from cyanobacterium
Oscilla-toria Planktothrix [21], which blocks sustained TLR4
stimulation, have been documented RSCL-0409,
gluco-disaccharide derivative, is one such initiative
capable of developing into a potential TLR antagonist
On the basis of our screening results, we short listed
RSCL-0409 for further studies With a 50% inhibitory
concentration of 10.6 lm, RSCL-0409 inhibited
LPS-stimulated TNF-a secretion from monocytic cells in a
dose-dependent manner LPS signalling is initiated by
its binding to LBP, followed by subsequent binding to
CD14, TLR4 and MD-2 complex on the cell surface
[22] However, depending on the LPS concentration, the
pathway may occur in an LBP⁄ CD14-dependent way or
in an independent way [23] Concentrations up to
100 ngÆmL)1 initiate LBP⁄ CD14 complex-dependent
binding and higher concentrations set up LBP⁄
CD14-independent signalling We have worked with varying
concentration of LPS (250–1000 ngÆmL)1) and
irrespec-tive of the LPS concentration, RSCL-0409 pretreated
cells inhibited TNF-a secretion up to 1000 ngÆmL)1,
making it a potential candidate for use in clinical
condi-tions such as septic shock, where LPS concentracondi-tions
are known to be very high The effects were similar in
PBMCs and RAW264.7 cells, indicating its effectiveness
on immune cells of different origins
Signalling through LPS induces the expression of many cytochemokines and adhesion molecules apart from TNF-a [6], with NF-jB being the prominent transcription factor involved in their regulation Our preliminary studies indicated that RSCL-0409 inhibited the mRNA expression of pro-inflammatory genes ICAM-1, Cox-2 and IL-8 Hence, we postulated that RSCL-0409 might suppress the activation of NF-jB,
as the above are regulated through it RSCL-0409 inhibited translocation into the nucleus and suppressed NF-jB-dependent reporter gene expression, indicating the inhibitory action of RSCL-0409 at the promoter level On the basis of these results, it appeared that RSCL-0409 acted upstream of IKK phosphorylation
In order to delineate the probable site of action of RSCL-0409, we decided to check the upstream acces-sory and adaptor molecules
LPS signalling through the activation of TLRs is very well established, with TLR4 largely implicated as the prominent mammalian LPS sensor [24] Simulta-neously, studies have also implicated TLR2 mediating LPS signalling in cells [25,26] Furthermore, Aderem & Ulevitch [27] postulated that in RAW cells, the oligo-merization of TLR receptors created functionally dis-tinct LPS-specific signalling receptors unlike the existing conventionally accepted CD14–TLR4 pathway involved in the activation of NF-jB leading to TNF-a expression in RAW cells [28], where they showed TLR6 and TLR2 co-operating in the signalling pro-cess In addition, there are documented reports that TLR2, -3, -4 and -9 recognize peptidoglycan, poly (I:C), LPS and CpG DNA, respectively [29–31]
In order to identify and delineate the probable mecha-nism of action of RSCL-0409, we studied the ability of various TLR ligands for cytokine TNF-a production in the presence of RSCL-0409 We were able to detect TNF-a secretion in cells stimulated with TLR1⁄ 2, TLR4 and TLR6 TLR3, -7, -8 and -9 are known to predomi-nantly secrete interferon-a [1] We speculate that it might be the reason for not having detectable levels of TNF-a following stimulation with these ligands in our system Analysing our data, we observed RSCL-0409’s selectivity to suppress TLR1⁄ 2- and TLR4-mediated TNF-a production in THP-1-stimulated cells Similar results were noted in human PBMCs However, we did not see any inhibitory effects on TNF-a released due to TLR6 ligand stimulation TLR1⁄ 2 ligand from the kit is Pam3CSK, a synthetic tripalymitoyl lipopeptide, which
is known to potentially activate monocytes and macro-phages [32] and TLR6 is a macrophage-stimulating lipopeptide-2 [33] known to activate the cells when it
0
500
1000
1500
***
***
2000
Contr
ol
Placebo
LPS (225 µg)
RSCL-0409 (10 mg·kg
–1 )
RSCL-0409 (20 mg·kg
–1 )
RSCL-0409 (10 mg·kg
RSCL-0409 (20 mg·kg
Fig 6 RSCL-0409 suppresses LPS-induced TNF-a release in
Balb ⁄ c mice Specific pathogen-free female Balb ⁄ c mice
(5–6 weeks, five animals per group) weighing 20–30 g were
intra-peritoneally injected with LPS (225 lgÆmL)1) with and without
pre-treatment of RSCL-0409 (10 and 20 mgÆkg)1) RSCL-0409 was
injected intraperitoneally 30 min before LPS injection One hour after
LPS injection, blood was collected retro-orbitally under anaesthesia
and serum analysed for TNF-a secretion ***P value < 0.001; LPS
treated vs RSCL-0409 treated Control represents untreated animals.
Trang 9heterodimerizes with TLR2 In addition, we have ruled
out any likely contamination in TLR2 agonist by
endo-toxin⁄ LPS by thorough assessment using the Limulus
amoebocyte lysate Therefore, it is plausible that
RSCL-0409 inhibits cytokine TNF-a production induced by an
additive process of TLR2 and TLR4 Corroborating
these data are the results observed in RAW cells, when
we evaluated the nitrite levels following stimulation with
TLR ligands Thus, the data suggest that RSCL-0409
suppresses the activation of cells by TLRs, probably an
upstream event in TLR1⁄ 2-, TLR4-mediated signalling,
and has the ability to recognize a lipopeptide
LPS signalling through TLRs involves four adaptor
molecules, MyD88, TIRAP, TRIF and TRAM [34]
Furthermore, two signalling pathways,
MyD88-depen-dent and MyD88-indepenMyD88-depen-dent pathways, have been
elu-cidated downstream of TLR2 and TLR4 [35–37] The
downstream signalling is quite complex and comprises
IRAK1–IRAK4–TNFR-associated factor 6 (TRAF6),
which transmits the signals to the TAK1 and IKK
com-plex [1] This activates the p65–p50 NF-jB heterodimer
complex, which translocates signals into the nucleus and
transcribes the NF-jB transcription factor, which
initi-ates the transcription of inflammatory mediators [38]
We checked the mRNA expression of the genes involved
in this signalling process following LPS stimulation with
and without pretreatment of RSCL-0409 Preliminary
data showed that RSCL-0409 downregulated TIRAP,
IRAK1 and IRAK4 mRNA levels together with TIRAP
and MyD88 protein levels (data not shown) TRAF6,
which did not show any visible upregulation upon LPS
stimulation, remained unaffected with RSCL-0409
pre-treatment Documented evidence shows that
recruit-ment of the adaptors involved in TLR signalling could
lead to the activation of multiple intracellular cascades,
including extracellular signal-regulated kinases, c-Jun
N-terminal kinases, p38 and NF-jB, involving their
phosphorylation [39,40] Therefore, we determined the
effect of RSCL-0409 on LPS-induced phosphorylation
of mitogen-activated protein kinases, extracellular
sig-nal-regulated kinase and p38 However, we did not
observe any effect on their phosphorylation
(unpub-lished data) There are reports of some antagonists, such
as 6-shogaol, the most bioactive component of ginger
[41], curcumin [42] and garlic extracts [43], inhibiting
TLR-mediated signalling by inhibiting the dimerization
of TLR4, a process that activates downstream signalling
pathways We are currently evaluating this possibility
In addition, we are also studying other possible targets,
such as the TLR4 coreceptor MD2, which directly binds
the disaccharide moiety of lipid A [44] Because cell
sig-nalling is a complex mechanism involving several
inter-related processes, we are not ruling out other possible
mechanisms of NF-jB- or TLR-mediated signalling inhibition
On the basis of the current evidence, we set forth to test RSCL-0409’s efficacy in an in vivo model TNF-a plays a pivotal role as a mediator of the host’s response
to LPS infection Therefore, blocking or antagonizing TNF-a in sepsis or a sepsis-like condition will definitely have immense therapeutic potential Our studies in Balb⁄ C in LPS-induced TNF-a secretion followed a pattern reported by many investigators, with TNF-a levels peaking around 60–90 min (unpublished data) However, pretreatment with RSCL-0409 at doses of 10 and 20 mgÆkg)1 significantly reduced TNF-a produc-tion, reconfirming our in vitro data Furthermore, treat-ment with RSCL-0409 alone or placebo did not lead to any detectable levels of TNF-a, implicating the specific-ity of the TNF-a inhibition due to RSCL-0409 How-ever, we would also like to point out that the LPS dose (225 lg mice)1) is similar to that given for the septic shock model Because we had observed that RSCL-0409 exhibited the ability to inhibit TNF-a secretion at LPS doses of 1 mgÆmL)1 we also looked at the effect of RSCL-0409 in preventing lethality induced by septic shock at two doses, 25 and 50 mgÆkg)1 We observed an
33 and 67% increase in survival ability in mice over a
30 h period (unpublished data) at respective doses In this context, our result has tremendous clinical applica-tion potential in attenuating LPS-mediated TNF-a,
a property useful in delaying the onset of sepsis
In conclusion, we have clearly demonstrated that RSCL-0409 prevents the expression of NF-jB-regulated genes in monocytes through a TLR-dependent process Preliminary data suggest that the inhibitory effect is exerted via the MyD88-dependent signalling cascade Investigations are continuing to elucidate the specific target molecule for RSCL-0409 RSCL-0409 is definitely
a good candidate and further studies evaluating its potential therapeutic applications are in progress
Materials and methods
Materials
RSCL-0409 (Fig 1A) was synthesized at Reliance Life Sciences (Navi Mumbai, Maharashtra, India) The synthesis was carried out by coupling glycosyl donor 2, 3-di-O-acetyl-4-O-chloroacetyl-6-O-levulinoyl-b-d-glucopyranosyl trichloroacetimidate with glycosyl acceptor 1-O-(p-methoxy-phenyl)-2-deoxy-2-trichloroacetamido-4,
purified using a silica gel column (40% ethyl acetate in
Trang 10crystallized in ethyl acetate⁄ hexane HPLC analysis of the
compound was dissolved in dimethylsulfoxide at a stock
Indi-vidual aliquots were used for carrying out the in vitro and
anhy-drous solvents purchased from commercial sources and used
without further purification Chromatographic separations
were performed on silica gel using the solvent system
indi-cated All reactions were monitored by TLC using precoated
silica gel plates (Merck, Whitehouse Station, NJ, USA)
Purity and yields were checked by chromatography and
spectroscopy
LPS (from E coli serotype O55: B5) was obtained from
Sigma-Aldrich (St Louis, MO, USA) Penicillin,
streptomy-cin, RPMI 1640 medium, sodium pyruvate and fetal bovine
serum were obtained from Gibco (part of Invitrogen,
Carls-bad, CA, USA) Tris, glycine, b-mercaptoethanol, glucose,
sodium bicarbonate, NaCl, SDS, BSA, LPS and MTT were
obtained from Sigma-Aldrich Polyclonal antibody anti-p65
was obtained from Cell Signaling Technology (Beverly,
MA, USA) Anti-rabbit secondary horseradish peroxidase
was obtained from Jackson Immuno Research (West
Grove, PA, USA) Trizol was obtained from Invitrogen
(Carlsbad, CA, USA) Chemiluminescence ECL was
pur-chased from Amersham (Arlington Heights, IL, USA) The
TNF-a Duo-Set ELISA detection kit was obtained from
R&D Systems; TLR ligands (1–9) were purchased from
Apotech (Enzo Life Sciences, Geneva, Switzerland) Quanti
Blue was purchased from Invivogen (San Diego, CA,
USA) PCR kits were obtained from Abgene (Epsom, UK)
The cDNA synthesis kit was obtained from ABI Systems
(Weiterstadt, Germany) All other reagents and chemicals
were purchased from Sigma, unless stated otherwise
Cells
The THP-1 promonocytic cell line and RAW264.7 cells
obtained from ATCC (Manassas, VA, USA) were cultured
in RPMI 1640 and DMEM, respectively, containing 10%
heat-inactivated fetal bovine serum supplemented with 1%
pur-chased from Invivogen, overexpress CD14 and were stably
transfected with a NF-jB inducible reporter plasmid system
expressing a SEAP The culture conditions were similar
to normal THP-1 cells with the addition of Zeocin and
Blasticidin antibiotics in the culture media Human PBMCs
were isolated from peripheral blood obtained from healthy
human volunteers (as per the procedure approved by the
appropriate regulatory committee) by density gradient
centrifugation using HistoPaque-1077 (Sigma-Aldrich) and
suspended in RPMI 1640 medium containing 10%
Treatment of the cells for quantification of TNF-a
well in 96-well culture plates and incubated overnight The cells in freshly replenished serum-free RPMI media were
24 h in the presence or absence of RSCL-0409 (50 lm) Culture supernatants were collected by spinning down the
further use The amount of secreted TNF-a in the super-natants was assayed using specific Duo-Set ELISA
manufacturer’s instructions For assays involving PBMCs and RAW264.7 cells, the 96-well plates were similarly
proto-col was followed for assaying TNF-a in these cells For all experiments, RSCL-0409 was dissolved in dimethylsulfox-ide, diluted with appropriate medium and added to the cells
1 h before any stimulation
Measurement of nitrite
RAW264.7 cells provide an excellent model for evaluations
of potential inhibitors on the pathway leading to the induc-tion of inducible nitric oxide synthase and NO producinduc-tion
NO production was determined in RAW264.7 cells from the National Center of Cell Science (NCCS, Pune, India) cultured in colour-free Dulbecco’s modified Eagle’s medium with standard supplements by measuring the amount of nitrite from the cell culture supernatant RAW264.7 cells
per well) were stimulated for 24 h with RSCL-0409
inter-feron-c for 24 h Nitrite was then measured using the Gri-ess reaction One hundred microlitres of cell culture supernatant was reacted with 100 lL Griess reagent fol-lowed by spectrophotometric measurement at 540 nm Nitrite concentrations in the supernatants were determined
by comparison with a sodium nitrite standard curve
Cell viability assay
Cell viability was assessed by morphology and by reduction
of the tetrazolium salt MTT by mitochondrial dehydrogen-ases, according to the manufacturer’s instructions (Sigma) Cytotoxicity studies were performed in 96-well plates
cells per well) were seeded