Báo cáo khoa học: Novel synthetic gluco-disaccharide RSCL-0409 – a lipopolysaccharide-induced Toll-like receptor-mediated signalling antagonist doc

Novel synthetic gluco-disaccharide RSCL-0409 –
a lipopolysaccharide-induced Toll-like receptor-mediated
signalling antagonist
Mani D. Kalluri1,*, Praneel Datla2,*, Akshaya Bellary1,*, Khalander Basha1, Ashwani Sharma1,
Anuradha Sharma1, Shiva Singh1, Shakti Upadhyay2 and Vikram Rajagopal1
1 Drug Discovery and Development Group, Reliance Life Sciences Pvt. Ltd, Navi Mumbai, India
2 EID Parry India Ltd, Chennai, India

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:
*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-glucopyranosyl]-(1-3)-1-O-(p-methoxyphenyl)-2-deoxy-2-N-trichloroacetyl-4,6-O-benzylidene-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 inflammation, where pretreatment with RSCL-0409 resulted in significant inhibition 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 antagonists and could definitely be a promising therapeutic agent for inflammatory diseases whose pathogenesis involves TLR2- or TLR4-mediated
signalling processes.

Introduction
Humans have evolutionarily conserved immune receptors, 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

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-trichloroacetyl4,6-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.

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RSCL-0409 inhibits LPS-induced TLR signalling

bacteria and viruses, triggering inflammatory and
antiviral responses that destroy the invading pathogens. So far, 11 TLRs have been described and for
most of them, except TLR10, natural ligands have
been identified. These include various proteins, lipopeptides, lipoteichoic acid, lipopolysaccharides (LPS)
and oligonucleotides (double-stranded RNA, singlestranded 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, activates monocytes and macrophages, leading to the production 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 tranducing 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 transferred to the TLR4–MD-2 complex [4,5]. Activation of
mitogen-activated protein kinases and nuclear factorjB (NF-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 autoimmune 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 pro1640

M. D. Kalluri et al.

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-acetyl6-O-levulinoyl-b-d-glucopyranosyl]-(1-3)-1-O-(p-methoxyphenyl)-2-deoxy-2-N-trichloroacetyl-4,6-O-benzylidene-a-d-glucopyranoside (RSCL-0409), from the
vast library of analogues synthesized, which selectively inhibits the TLR-mediated production of

TNF-a and also mRNA expression of various proinflammatory 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 ngỈmL)1 LPS in the presence and absence of various 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 (calculated using biodatafit software). We observed similar
inhibition in both RAW264.7 cells and PBMCs stimulated 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 ngỈmL)1 LPS 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-dimethylthiazol-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 inhibitory effect on the production of the pro-inflammatory

mediator TNF-a from both mouse and human
monocytes and macrophages stimulated with LPS.

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M. D. Kalluri et al.

RSCL-0409 inhibits LPS-induced TLR signalling

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.

Inhibitory 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 pretreated 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 (unpublished 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)

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RSCL-0409 inhibits LPS-induced TLR signalling

M. D. Kalluri et al.

A 2500

B 3500
NS

3000
2500

1500

***

TNF (pg/ml)

TNF (pg/ml)

2000

1000
500

***

***

2000
1500
1000

***
500

LP
S
L04
09
SC
R

R

SC

+

L04
09
R

SC

C

on
tro
l

LP
S

0

L04
09

C

LP
(1

S
,1
0,
50
R
,1
SC
00
L04
µM
09
)
(1
R
µM
SC
)+
L04
LP
09
S
(1
0
R
µM
SC
)+
L04
LP
09

S
(5
R
0
SC
µM
L)+
04
LP
09
S
(1
00
µM
)+
LP
S

on
tro
l

0

C 3000

D

125

NS

2500

*

1500

***

1000

***

500

% Cell viability

TNF-α (pg/ml)

100
2000

75
50

25
S

µM

)
00

µM
)
0

0

LSC
R

R

SC

L-

04

04

09

09

(1

(5

(1
09
04
LSC

R

R

L-

SC

L-

04

04

09

09

(1

(1

µM
)

µM
)

ls
el

00

C

µM
)+

LP

LP

S

S
µM
)+
R

R

SC

SC

SC

L-

L-

04

04

09

09

(5

(1

0

0

µM
)+

µM
)+

LP

LP

S

µM
)
R

0

R

SC

L-

R

04

SC

09

L-

(1

04

,1

09

0,

(1

50

,1

C

00

on

LP

tro

l

S

0

E

1600
250 ng·mL–1

1400

500 ng·mL–1
1000 ng·mL–1

TNF (pg/ml)

1200
1000
800
600
400
200

S

µM

R

SC

L04

09

+

LP

S
0
10

50

µM

R

SC

L04

09

+

LP

S
LP
+
09
L04
SC
R

µM
10

1

µM

R

SC

L04

09

+

LP

LP

S

S

0

Fig. 2. RSCL-0409 suppression of LPS-induced TNF-a in a dose-dependent manner and in different cell lines. Serum-starved human monocytic 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 · 105 cellsỈwell)1) were pretreated with RSCL-0409 in increasing concentrations (1, 10, 50
and 100 lM) 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 ngỈmL)1) with and without pretreatment with
RSCL-0409 (1–100 lM) for 24 h. The supernatant was then assayed for the ability of the cells to release TNF-a by ELISA. The data are a representation of two independent experiments.

1642

FEBS Journal 277 (2010) 1639–1652 ª 2010 Reliance Life Sciences Pvt Ltd. Journal compilation ª 2010 FEBS


M. D. Kalluri et al.

A

RSCL-0409 inhibits LPS-induced TLR signalling

Effect of RSCL-0409 on various TLR ligands in THP-1 cells

B 1400

Effect of RSCL-0409 on various TLR ligands in PBMCs

3000
Ligands alone
RSCL-0409

1200

NS

1000
TNF (pg/ml)

2000
1500
1000

**

NS

200

***

0

0

35
NS

30

Ligands alone
RSCL-0409

D 24.0

20.0

NS

20
15

NS

NS

10

*

5

Nitrite (µM)

25

*

16.0
12.0

***

8.0

***

4.0

***

0

***
S
0

µM
)+

LP
(1
09
04
R

SC

L-

LSC
R

LP

S

S
µM
)+
(5
09
04

09
LSC
R

R

SC

L-

04

04

09

C

(1

(2

.2

pG

5

.5

µM
)+

µM
)+

LP

LP

S

S

tro
on

N
O

D

l

95
23

48
R

-8

2
pal

LP

)

S
M

SK
3C

I:C

C
3H
4.

on
C
Pa
m

Po
ly
(

tro

l

l

0.0
C

Nitrite (µM)

**

600
400

500

C

Ligands alone

RSCL-0409

**

800

LP

TNF (pg/ml)

2500

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 · 105 cellsỈwell)1, were pretreated with RSCL-0409 (50 lM) 1 h before TLR ligand treatment. The pretreated cells were stimulated with various TLR ligands at different concentrations (TLR1 ⁄ 2 75 ngỈmL)1, TLR3 75 lgỈmL)1, TLR4 750 ngỈmL)1, TLR5 75 ngỈmL)1,
TLR6 75 ngỈmL)1, TLR7 ⁄ 8 7.5 lgỈmL)1 and TLR9 7.5 lgỈmL)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 experiments. *P value < 0.05, **P value < 0.01, ***P value < 0.001; ligand-treated cells vs RSCL-0409-treated cells, NS, not significant.

induced by TLR ligands in RAW264.7 cells. RSCL0409 selectively inhibited TLR1 ⁄ 2- and LPS-induced
NO production (Fig. 3C). RSCL-0409 had no significant 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 RSCL0409 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 THP1 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 stimulation, respectively. Furthermore, we wanted to determine 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 degradation of IjB, allowing the free NF-jB to translo-

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RSCL-0409 inhibits LPS-induced TLR signalling

M. D. Kalluri et al.

A 16.0

B 9.0
TNF-α

IL-6
8.0

12.0

7.0
6.0

10.0
Fold change

Fold change

14.0

8.0
6.0

5.0
4.0
3.0
NS

4.0
2.0
2.0

***

NS
0.0
Cell control

Cells + LPS

Cells + RSCL0409 (50 µM)

0.0

Cells + RSCL0409 (50 µM)
+ LPS

C

***

1.0
Cell control

Cells + RSCL0409 (50 µM)

Cells + LPS

Cells + RSCL0409 (50 µM) + LPS

RSCL-0409 + LPS
LPS

RSCL-0409
Control

Lanes

M

1

2

3

4

Time (min)

60

60

60

60

LPS

–

–

+

+

RSCL-0409

–

+

–

+

0

1

2

3

ICAM-1

4
5
Fold change

6

7

8

RSCL-0409 + LPS

LPS

ICAM-1

282 bp

Cox-2

278 bp

RSCL-0409

Control

0

1

2

Cox-2

IL-8

221 bp

β-actin

3
Fold change

4

5

6

313 bp

RSCL-0409 + LPS
LPS
RSCL-0409
Control
0
IL- 8

2

4
6
Fold change

8

10

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 exposure to LPS (250 ngỈmL)1) with or without 50 lM 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 correction 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.

cate into the nucleus to activate genes with NF-jBbinding regions. Therefore, we checked whether
RSCL-0409 blocked any signals responsible for NF-jB
1644

signalling leading to nuclear translocation. RSCL-0409
prevented IjB-a degradation and phosphorylation of
the p65 subunit (data not shown), resulting in the

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M. D. Kalluri et al.

A

Tubulin
Lanes

Discussion
The release of multiple pro-inflammatory cytochemokines 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 signalling mechanism needs to modulate this pro-inflammatory 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 identified 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

2

3

4

5

6

7

0

30

60

120

30

60

120

LPS

–

+

+

+

+

+

+

RSCL-0409

–

–

–

–

+

+

+

RSCL-0409+LPS-60
RSCL-0409+LPS-30
RSCL-0409+LPS-15
LPS-120
LPS-60
LPS-30
Control
0

1

2

3

4

5

6

7

8

9

Fold change

120.0
100.0

NS

80.0

**

60.0

***

40.0
NS

20.0

NS

NS

S

04

04

09

09

(5

(1

0

0

µM

µM

)+

)+

LP

LP

LP

S

S

)

L-

L-

SC
R

R

+
C

el

ls

ls
el

C

C

el

ls

+

+

C

R

SC

SC

+

L-

R

04

SC

09

L-

(1

04

µM

09

)+

(5

(1

0

0

µM

µM

)
µM
09
el

ls

ls
el
C

C

el

ls

+

+

R

R

SC

SC

L-

L-

04

04

el

09

lc

(1

on

tr

LP

ol

S

)

0.0

C

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 treated 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 mgỈkg)1 injected RSCL-0409, respectively.

1

Time (min)

B

Effect of RSCL-0409 on LPS-induced TNF-a
production in vivo

p65NF-kB

Relative SEAP secretion

inhibition of downstream signalling. Further downstream, we saw partial translocation of NF-jB from
the cytoplasm into the nucleus within 60 min, which
was blocked when cells were pretreated with RSCL0409, 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 pretreatment 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.

RSCL-0409 inhibits LPS-induced TLR signalling

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 lM 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 promoter, were stimulated with LPS (250 ngỈmL)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 independent 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.

that RSCL-0409 exerts similar inhibitory effects in
both human monocytes and mouse macrophages, indicating that its potency is spread across different
species.
With TLRs gaining prominence as drug targets,
numerous agonists are currently being developed [15].

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RSCL-0409 inhibits LPS-induced TLR signalling

M. D. Kalluri et al.

TNF release (pg/ml)

2000
1500
1000

***

***

500

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R

SC
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04
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kg
09
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04
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R
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)
04
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S
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C
on
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o

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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 intraperitoneally injected with LPS (225 lgỈmL)1) with and without pretreatment of RSCL-0409 (10 and 20 mgỈkg)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.

The development of antagonists has gained impetus
following concerns that agonists have not been effective in specific targeting. Currently, TAK-242 [16] and
E-5564 [17], apart from the few reported lipid A analogues as antagonists [18–20], are in the last phase of
clinical developmental. Furthermore, reports of LPSlike molecules extracted from cyanobacterium Oscillatoria 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 LPSstimulated 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 ngỈmL)1 initiate LBP ⁄ CD14 complex-dependent
binding and higher concentrations set up LBP ⁄ CD14independent signalling. We have worked with varying
concentration of LPS (250–1000 ngỈmL)1) and irrespective of the LPS concentration, RSCL-0409 pretreated
cells inhibited TNF-a secretion up to 1000 ngỈmL)1,
making it a potential candidate for use in clinical conditions such as septic shock, where LPS concentrations
are known to be very high. The effects were similar in
1646

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 accessory 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]. Simultaneously, studies have also implicated TLR2 mediating
LPS signalling in cells [25,26]. Furthermore, Aderem &
Ulevitch [27] postulated that in RAW cells, the oligomerization of TLR receptors created functionally distinct 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 process. 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 mechanism 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 predominantly 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 macrophages [32] and TLR6 is a macrophage-stimulating
lipopeptide-2 [33] known to activate the cells when it

FEBS Journal 277 (2010) 1639–1652 ª 2010 Reliance Life Sciences Pvt Ltd. Journal compilation ª 2010 FEBS


M. D. Kalluri et al.

heterodimerizes with TLR2. In addition, we have ruled
out any likely contamination in TLR2 agonist by endotoxin ⁄ LPS by thorough assessment using the Limulus
amoebocyte lysate. Therefore, it is plausible that RSCL0409 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-dependent and MyD88-independent pathways, have been elucidated 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 complex [1]. This activates the p65–p50 NF-jB heterodimer
complex, which translocates signals into the nucleus and
transcribes the NF-jB transcription factor, which initiates 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 pretreatment. Documented evidence shows that recruitment 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 signal-regulated kinase and p38. However, we did not
observe any effect on their phosphorylation (unpublished 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 signalling is a complex mechanism involving several interrelated processes, we are not ruling out other possible

RSCL-0409 inhibits LPS-induced TLR signalling

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 mgỈkg)1 significantly reduced TNF-a production, reconfirming our in vitro data. Furthermore, treatment with RSCL-0409 alone or placebo did not lead to
any detectable levels of TNF-a, implicating the specificity of the TNF-a inhibition due to RSCL-0409. However, 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 mgỈmL)1 we also looked at the effect of
RSCL-0409 in preventing lethality induced by septic
shock at two doses, 25 and 50 mgỈkg)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 application 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-Oacetyl-4-O-chloroacetyl-6-O-levulinoyl-b-d-glucopyranosyl
trichloroacetimidate with glycosyl acceptor 1-O-(p-methoxyphenyl)-2-deoxy-2-trichloroacetamido-4, 6-O-benzylidene-ad-glucopyranose using Lewis acid. The crude product was
purified using a silica gel column (40% ethyl acetate in hexane) to give disaccharide RSCL-0409 (91%) Rf 0.62 (50%
ethyl acetate ⁄ hexane). The column-purified RSCL-0409 was

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1647


RSCL-0409 inhibits LPS-induced TLR signalling

crystallized in ethyl acetate ⁄ hexane. HPLC analysis of the
purified compound showed  98.5% purity (Fig. 1B). The
compound was dissolved in dimethylsulfoxide at a stock concentration of 10 mm, aliquoted and stored at )20 °C. Individual aliquots were used for carrying out the in vitro and
in vivo assays. All procedures were carried out using anhydrous solvents purchased from commercial sources and used
without further purification. Chromatographic separations
were performed on silica gel using the solvent system indicated. 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, streptomycin, RPMI 1640 medium, sodium pyruvate and fetal bovine
serum were obtained from Gibco (part of Invitrogen, Carlsbad, 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 purchased 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%
penicillin ⁄ streptomycin, as suggested by ATCC, in a 5%
CO2 humid atmosphere at 37 °C. THP-1 blue CD14, purchased 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% heatinactivated fetal bovine serum, 100 mL)1 penicillin G
and 100 lgỈmL)1 streptomycin.

1648

M. D. Kalluri et al.

Treatment of the cells for quantification of TNF-a
THP-1 cells were plated at a density of 2 · 105 cells per
well in 96-well culture plates and incubated overnight. The
cells in freshly replenished serum-free RPMI media were
stimulated
with
various
TLR
ligands
[TLR1 ⁄ 2
(Poly I:C)
(Pam3CSK4.3HCl)
75 ngỈmL)1, TLR3
75 lgỈmL)1, TLR4 (LPS from E. coli) 750 ngỈmL)1, TLR5
(purified flagellin) 75 ngỈmL)1, TLR6 (Malp-2) 75 ngỈmL)1,
TLR7 ⁄ 8 [Poly(U) potassium salt] 7.5 lgỈmL)1 and TLR9
(CpG ODN 2395) 7.5 lgỈmL)1], LPS (250 ngỈmL)1) for
24 h in the presence or absence of RSCL-0409 (50 lm).
Culture supernatants were collected by spinning down the

cells and the medium collected and stored at )80 °C until
further use. The amount of secreted TNF-a in the supernatants was assayed using specific Duo-Set ELISA development systems (R&D Systems) according to the
manufacturer’s instructions. For assays involving PBMCs
and RAW264.7 cells, the 96-well plates were similarly
seeded at a density of 2 · 105 cellsỈwell)1. A similar protocol was followed for assaying TNF-a in these cells. For all
experiments, RSCL-0409 was dissolved in dimethylsulfoxide, 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 induction of inducible nitric oxide synthase and NO production.
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
(5 · 104 per well) were stimulated for 24 h with RSCL-0409
and ligands alone for TLRs, 75 ngỈmL)1 Pam3CSK4 for
TLR2 ⁄ TLR1, 75 lgỈmL)1 poly (I:C) for TLR3,
250 ngỈmL)1 LPS for TLR4, 75 ngỈmL)1 Malp-2 for TLR6,
10 lm R-848 for TLR7 ⁄ 8 and 7.5 lgỈmL)1 CpG ODN
2395 for TLR9 in the presence of 0.1 ngỈmL)1 mouse interferon-c for 24 h. Nitrite was then measured using the Griess reaction. One hundred microlitres of cell culture
supernatant was reacted with 100 lL Griess reagent followed 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 dehydrogenases, according to the manufacturer’s instructions (Sigma).
Cytotoxicity studies were performed in 96-well plates.

THP-1 cells (2 · 105 cells per well) were seeded. RSCL-

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M. D. Kalluri et al.

RSCL-0409 inhibits LPS-induced TLR signalling

0409 (1–100 lm) was added to the cells, and the plates were
incubated for 24 h. The cells were then washed once before
adding 50 lL fetal bovine serum-free medium containing
MTT (5 mgỈmL)1). After 4 h of incubation at 37 °C, the
medium was discarded and the formazan blue that formed
in the cells was dissolved in dimethylsulfoxide. The absorbance was measured at 570 nm using a powerwave Xs from
Biotek Instruments (Winooski, VT, USA).

p65NF-jB bands was normalized by tubulin. The bands
obtained were quantitated using imagej software version
1.42.

Real-time quantitative PCR analysis of TNF-a and
IL-6 expression

Nuclear extracts were prepared from THP-1 cells according
to the modified procedure of Dignam et al. [45]. Briefly,
3 · 106 cells were pretreated with RSCL-0409 for 1 h followed by LPS treatment for 30, 60 and 120 min. Following
incubation, the cells were lysed in 400 lL lysis buffer (10 mm
Hepes pH 7.9, 100 mm KCl, 1.5 mm MgCl2, 0.1 mm EGTA,
0.5 mm dithiothreitol, 0.5 mm phenylmethanesulfonyl fluoride, 0.5% Nonidet P-40 and 1 lLỈmL)1 protease inhibitor

cocktail; Calbiochem, La Jolla, CA, USA). The homogenate
was centrifuged in a microfuge at 10 000 g for 5 min. The
supernatant was discarded, and the nuclear pellet was resuspended in 50 lL nuclear extraction buffer (10 mm Hepes pH
7.9, 1.5 mm MgCl2, 420 mm NaCl, 0.1 mm EGTA, 0.5 mm
dithiothreitol, 5% glycerol, 0.5 mm phenylmethanesulfonyl
fluoride and 1 lLỈmL)1 protease inhibitor cocktail) with
intermittent mixing in ice. The nuclear extract was obtained
by centrifuging at 10 000 g for 10 min at 4 °C. The concentration of protein in each sample was then quantified using
the Bradford method (Bio-Rad, Hercules, CA, USA).
Western blot analysis was carried out to check for p65 levels
in the extracts.

THP-1 cells seeded at a density of 3 · 106 cellsỈwell)1 in a
six-well plate were stimulated with 250 ngỈmL)1 LPS in the
presence or absence of RSCL-0409 (50 lm) for 1 h. Total
RNA was isolated from these cells and cDNA was synthesized. LPS-treated cells acted as a positive control. All
quantitative real-time PCR (TaqManÔ) primers and probes
were obtained from Applied Biosystems (Weiterstadt, Germany). For the detection of TNF-a, IL-6 and internal control b-actin predeveloped assay reagents were used. The
PCR was performed utilizing 1 lL cDNA per reaction in
triplicates of 25 lL volume on an ABI 7500 real-time PCR
machine using a two-step PCR protocol after the initial
denaturing of the cDNA (10 min at 95 °C) with 40 cycles
of 95 °C for 15 s and 60 °C for 1 min. The universal
master mix, as obtained from Applied Biosystems,
included all reagents, including Taq-polymerase, apart
from specific primers and probes. All amplification
batches included no template controls. Quantitation of
mRNA was performed using the comparative threshold
cycle method. The highest control level attained by the
stimulation (without RSCL-0409) was regarded as 100%,

and the levels of the control group at other time points
and the RSCL-0409-added group were expressed as a percentage of the highest control level. Data were analysed
using standard software.

Western blot analysis

RT-PCR analysis

Preparation of nuclear extracts

6

)1

THP-1 cells at 1 · 10 cellsỈmL
in serum-free RPMI
1640 medium were incubated with LPS (250 ngỈmL)1) for
different time points. Where indicated, THP-1 monocytes
were incubated with RSCL-0409 for 60 min before LPS
treatment. Cells were lysed following incubation in RIPA
buffer (1· phosphate-buffered saline, 1% Nonidet P-40,
0.5% sodium deoxycholate, 0.1% SDS, 1 mm sodium
orthovanadate, 10 lgỈmL phenylmethanesulfonyl fluoride
and 1 lLỈmL)1 protease inhibitor cocktail). The protein
concentration in each sample was quantified using the
Bradford method. Twenty-five micrograms of protein were
size fractionated in a 10% SDS ⁄ PAGE gel and transferred
to a nitrocellulose membrane (BioTraceNT, Pall Corporation, Port Washington, NY, USA). Blots were probed
with an anti-p65 IgG (Cell Signaling Technology). Horseradish peroxidase-conjugated secondary antibodies were
used to develop the membrane and visualization of the

bands was performed using a chemiluminescent substrate
(Amersham). Blots were stripped and reprobed with tubulin to normalize the protein loading. Quantification of

THP-1 cells (3 · 106 cells) were seeded in a six-well dish.
The cells were treated with RSCL-0409 (50 lm) for 1 h followed by incubation with or without 250 ngỈmL)1 LPS.
After two washes with ice-cold phosphate-buffered saline,
the cells were harvested and total cellular RNA was isolated using TRIZOL Reagent (Invitrogen) according to the
manufacturer’s instructions. cDNA synthesis was carried
out using a high capacity cDNA reverse transcription kit
(ABI Systems). Amplification of ICAM-1, Cox-2 and IL-8
genes from the cDNA was carried out using the respective
gene-specific primers:
ICAM-1
5¢- CTGATGGGCAGTCAACAGCTAAAA - 3¢(sense)
5¢- TCCAGTTCAGTGCGGCACGAGAA - 3¢ (antisense)
Cox-2
5¢-ATGAGATTGTGGGAAAATTGCT- 3¢ (sense)
5¢- GGTAGATCATCTCTGCCTGAGTATC - 3¢ (antisense),
IL-8
5¢- GCCAAGGAGTGCTAAAGAACTTAG -3¢ (sense)

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1649


RSCL-0409 inhibits LPS-induced TLR signalling

5¢- GAATTCTCAGCCCTCTTCAAAAAC -3¢ (antisense)
b-actin, an internal control, was also amplified using the

following primers:
5¢- TCC TCC CTG GAG AAG AGC TA - 3¢ (sense)
5¢- AGT ACT TGC GCT CAG GAG GAC – 3¢ (antisense)
Furthermore, the TLR-related genes (TIRAP, IRAK-1,
IRAK-4 and TRAF6) were amplified from the cDNA
extracted from a similar experimental set using the respective gene-specific primers [46].
The amplified PCR products were then analysed on a
2% agarose gel electrophoresis and visualized under UV
with ethidium bromide. Quantification of bands for the
specific genes was normalized by b-actin.

CA, USA). The Neuman–Keuls test was used for multiple comparisons. Values of P < 0.05 were considered
as significant.

Acknowledgements
The authors gratefully acknowledge the encouragement
and support of Reliance Life Sciences Pvt Ltd, in carrying out the research work. PD and SU were part of
Reliance Life Sciences Pvt Ltd when the work was
undertaken. Their current affiliation is indicative of
their current address only, and not to be understood
as collaborative work.

References

Quanti Blue assay
The Quanti Blue assay is a reporter-based assay system to
determine the SEAP activity in supernatants of cell cultures
induced by NF-jB activators such as LPS. The assay was
carried out as per the manufacturer’s instructions. Briefly,
THP-1 CD14 blue cells were plated and cultured overnight

in 96-well plates (2 · 105 cellsỈwell)1). These cells were stimulated with LPS in the presence and absence of RSCL-0409
(50 lm). Culture supernatants were collected 24 h after LPS
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In vivo experiments: LPS-induced TNF-a release
in Balb ⁄ c mice
Specific pathogen-free female Balb ⁄ c mice (5–6 weeks)
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studies.

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