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Ursodeoxycholic acid inhibits colonic
mucosal cytokine release and prevents
colitis in a mouse model of disease
Joseph BJ Ward1, Orlaith Kelly1,2, Siobhan Smith3, Joan Ní Gabhann3, Murtaza Tambuwala4, Frank Murray2, Caroline
Jefferies3, Cormac Taylor4 and Stephen Keely1.
1RCSI, Beaumont Hospital. 2 Dept. of Gastroenterology, Beaumont Hospital. 3 RCSI, St. Stephens Green, 4 UCD.
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*
**
0
***
0
1
2
3
4
5
6
Time (days)
B
C
UDCA attenuates TLR-3 driven cytokine
release from colonic epithelial cells
Colon length (mm)
Vehicle
UDCA (30 mg/kg)
DSS (2.5%)
+ Vehicle
120
DSS (2.5%) + UDCA
(30 mg/kg)
***
80
*
60
40
20
0
***
Vehicle + UDCA Vehicle + UDCA
(30 mg/kg)
(30 mg/kg)
80
**
#
60
n = 6 - 12, ANOVA + Tukey
* compared to DSS + Veh
##
D
40
Untreated
DSS
Untreated
1.0
0.8
0.6
0.4
***
-
1
50
100
200
+
+
-
+
+
-
10
Control PBS
DSS (2.5 %)
DSS + 6MUDCA (30 mg/kg)
8
6
4
2
0
0
+
+
1
2
3
4
5
6
Time (days)
n=3-9
UDCA metabolites are elevated in a mouse
model of disease
+ DSS (2.5 %)
40
DSS
[UDCA] ( M)
n = 6,
+
-
12
Figure 5. 6MUDCA attenuates in vitro cytokine release, but is not protective in a
murine model of colitis. A) The TLR3 agonist, Poly I:C (25 μg/ml), significantly
increased TNF-a release compared to control. This response was reduced by cotreatment with either UDCA (200 mM) or a stable analogue, 6MUDCA (n = 5 p 
0.001). B) However, in contrast to UDCA, intraperitoneal administration of
6MUDCA (30mg/kg) to male C57 BL/6 mice receiving DSS in their drinking water,
did not reduce the DAI (n = 3 - 9).
PBS
Control P.IC
***
0.2
20
0
B
1.2
0.0
PI:C
UDCA
6MUDCA
*UDCA compared to DSS
RESULTS
[TNF- ] (% Poly I:C)
***
n = 6-12
Statistical analyses were performed using ANOVA and SNK or Tukey post hoc test.
100
***
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METHODS
A
A
6
2
6MUDCA
Disease activity index
Here we sought to investigate a potential role for UDCA in regulating
colonic epithelial cytokine release and to investigate the therapeutic
potential of UDCA in a mouse model of disease.
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Vehicle
UDCA (30 mg/kg)
UDCA (100 mg/kg)
DSS (2.5 %)
DSS + UDCA (30mg/kg)
DSS + UDCA (100mg/kg)
[bile acid] ( M)
A
12
Metabolically stable analogues of UDCA
are ineffective against colitis in a mouse
model of disease
TNF release
(fold change over PI:C)
Toll-like receptors (TLRs) play a critical role in innate immune
responses to intestinal pathogens. In inflammatory bowel disease
(Ulcerative Colitis and Crohn’s Disease), epithelial TLR expression is
increased. Activation of these TLRs is likely to play an integral role in
the pro-inflammatory cytokine release responsible for the large-scale
epithelial damage that is typical of active colitis. Ursodeoxycholic acid
(UDCA), a bile acid, is a well-established therapy for inflammatory
diseases of the liver, where it is known to act at least partly, through
inhibition of cytokine release. However, the role of UDCA in regulating
intestinal epithelial cytokine secretion is unknown.
UDCA ameliorates colitis in a mouse
model of disease
Disease Activity Index
INTRODUCTION
* compared to control
UDCA
CDCA
LCA
3- 7-oxo
***
30
***
***
20
**
10
# compared to Poly I:C
***
n = 4, ANOVA + SNK
1200 * compared to control
#
pg/ml
20
10
800
40
400
200
UDCA
PIC
0
-
+
-
D
+
+
+
***
* compared to control
200
+
+
+
n = 4, ANOVA + SNK
***
* compared to control
#
# compared to PIC
**
pg/ml
pg/ml
2.0
**
300
100
UDCA
PIC
+
-
IL-1
2.5
n = 4, ANOVA + SNK
0
-
E
IL-6
400
0
UDCA
PIC
***
30
20
* compared to 0 mg/kg vehicle
# compared to 0 mg/kg DSS
10
0
30
Vehicle + UDCA
(mg/kg)
1.0
#
***
0
1.5
0
30
DSS (2.5%) + UDCA
(mg/kg)
0.5
-
+
-
+
0.0
UDCA
PIC
+
+
-
+
-
+
+
+
Figure 1. UDCA attenuates TLR-3 driven cytokine release from T84 cells. A) The
TLR3 agonist, Poly I:C (25 μg/ml), significantly increased TNF-a release compared
to control, which was significantly reduced in a concentration-dependent manner
by co-treatment with UDCA (0-200 mM). Poly I: C-stimulated B) TNF-a release, C)
IL-8 release, D) IL-6 release and E) IL-1b release were all significantly reduced to
by co-treatment with UDCA (200 mM).
UDCA attenuates TRIF/TBK1 signalling
Effect of UDCA on P125 promoter transcription
Luciferase activity
(fold change over control EV)
Figure 6. UDCA administration alters the caecal bile acid pool. Intraperitoneal
administration of UDCA (30 mg/kg) to male C57 BL/6 mice receiving DSS in their
drinking water, significantly elevated levels of UDCA metabolites, LCA and 7keto LCA.
E
600
100
Control
UDCA (200 M)
*
80
Figure 3. UDCA is protective in a murine model of colitis. A)
Intraperitoneal administration of UDCA (30 mg/kg and 100 mg/kg) to
male C57 BL/6 mice receiving DSS in their drinking water, significantly
reduced the disease activity index (DAI) from 10 ± 0.3 (DSS alone) to 7.2 ±
0.7 (UDCA 30mg/kg) and 5.8 ± 0.5 (UDCA 100mg/kg) (n = 6 - 12, p 
0.001). B) Mice treated with DSS alone had shorter colons and a lack of
faecal pellet formation. In contrast, mice co-treated with UDCA had
longer colons with clear evidence of faecal pellet formation. C) The
average length of colons in DSS-treated mice was shorter than in mice cotreated with UDCA (30 mg/kg) (n = 6-12, p < 0.05). D) H and E staining was
performed on colonic sections and E) UDCA (30 mg/kg) significantly
reduced the inflammation score of the sections from 37.3 ± 0.3 in DSS
alone, to 29.0 ± 3.5 (ANOVA, n = 5, p  0.05).
Biochemistry of UDCA metabolism
ANOVA + SNK,
N = 6, p 0.05
60
40
20
0
EV
TRIF
TBK1
IKKe
IRF3
+ P125 luciferase
Figure 2. UDCA inhibits TLR signalling at TRIF/TBK1. Overexpression analysis
of proteins in the TLR signalling pathway revealed that UDCA (200 mM) exerts
its inhibitory effect at the TIR-domain-containing adapter-inducing interferonβ (TRIF)/TANK-binding kinase 1 (TBK1) junction (n = 6, p  0.05).
DSS
DSS
Basal + UDCA
n = 3, ANOVA + SNK,
** P 0.01, ** P 0.001
***
1000
UDCA
* vs respective basal bile acid
#
# compared to PIC
***
Basal
Figure 4. UDCA and its derivatives can be readily interconverted.
Liver enzymes along with bacterial enzymes in the colon can readily
interconvert UDCA to LCA and 7-keto LCA.
The UDCA metabolite, LCA prevents colitis
in a mouse model of disease
1.2
1.0
0.8
0.6
0.4
***
0.2
0.0
Poly I:C
0
-
0
+
0.001 0.1
+
+
1
+
LCA ( M; 24 hours)
n = 7, ANOVA + SNK,
p 0.001 compared to Poly I:C
10
+
Disease Activity Index
30
1400
***
IL-8
TNF release
(fold change over control)
n = 4, ANOVA + SNK
* compared to control
# compared to PIC
0
Inflammation score
40
pg/ml
C
TNF-
UDCA
B
12
Control
LCA (30 mg/kg)
DSS
DSS + LCA (30 mg/kg)
10
8
***
6
***
4
2
0
0
1
2
3
4
5
Time (days)
n = 5, * DSS + LCAcompared to DSS
Figure 7. LCA attenuates in vitro cytokine release and is protective in a murine
model of colitis. A) The TLR3 agonist, Poly I:C (25 μg/ml), significantly increased
TNF-a release compared to control. This response was reduced by co-treatment
with LCA (10 mM) (n = 7 p  0.001). B) Intraperitoneal administration of the UDCA
metabolite, LCA (30 mg/kg), to male C57 BL/6 mice receiving DSS in their drinking
water, significantly reduced the DAI from 11.2 ± 0.6 (DSS alone) to 5.4 ± 0.9 (n =
5, p  0.001).
SUMMARY & CONCLUSION
These studies reveal a novel role for UDCA in regulating colonic
epithelial cytokine secretion. UDCA significantly attenuates TLR-3dependent cytokine release from colonic epithelial cells through a
pathway dependent on TRIF/TBK-1. UDCA also ameliorates the
effects of colitis in a mouse model. Interestingly, metabolically stable
analogues of UDCA do not prevent colitis in a mouse model, whereas
the metabolic product of UDCA, LCA, practically abolishes DSSinduced colitis. In conclusion, our data suggest that, by virtue of their
effects in preventing TLR-induced proinflammatory
cytokine release, UDCA and its metabolic products
are good targets for developing new approaches to
treat IBD.
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