LCA (M; 24 hours)

TN

F

rele

as

e(fo

ld c

ha

ng

e o

ver c

on

trol)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 0 0.001 0.1 1 10

Poly I:C - + + + + +

n = 7, ANOVA + SNK,p 0.001 compared to Poly I:C

***

Ursodeoxycholic acid inhibits colonic mucosal cytokine release and prevents colitis in a mouse model of diseaseJoseph 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.

INTRODUCTIONToll-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.

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.

These studies reveal a novel role for UDCA in regulating colonic epithelial cytokine secretion. UDCA significantly attenuates TLR-3-dependent 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 DSS-induced colitis. In conclusion, our data suggest that, by virtue of their effects in preventing TLR-induced proinflammatorycytokine release, UDCA and its metabolic products are good targets for developing new approaches to treat IBD.

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).

A

0 30 0 30

Inflam

matio

n sco

re

0

10

20

30

40

Vehicle + UDCA(mg/kg)

DSS (2.5%) + UDCA(mg/kg)

***

***#

* compared to 0 mg/kg vehicle# compared to 0 mg/kg DSS

Effect of UDCA on P125 promoter transcription

EV TRIF TBK1 IKKe IRF3

Lu

ciferase activity

(fold

chan

ge o

ver co

ntro

l EV

)

0

20

40

60

80

100Control UDCA (200 M)

+ P125 luciferase

*

ANOVA + SNK, N = 6, p 0.05

Metabolically stable analogues of UDCA are ineffective against colitis in a mouse

model of disease

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 co-treatment 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).

B

Untreated DSS Untreated DSS

UD

CA

PBS

D

E

Biochemistry of UDCA metabolism

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.

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 interferon-β (TRIF)/TANK-binding kinase 1 (TBK1) junction (n = 6, p £ 0.05).

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 co-treated 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).

TN

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I:C)

0.0

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PI:C - - - + + +UDCA - + - - + -6MUDCA - - + - - +

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A

Control P.IC 1 50 100 200

[TN

F-

] (%

Po

ly I:C

)

0

20

40

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80

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120

***

[UDCA] (M)

***

** #

##

n = 6, * compared to control # compared to Poly I:C

A

UDCA attenuates TRIF/TBK1 signalling

METHODS

RESULTSUDCA attenuates TLR-3 driven cytokine

release from colonic epithelial cells

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).

Statistical analyses were performed using ANOVA and SNK or Tukey post hoc test.

Time (days)

0 1 2 3 4 5 6

Disease A

ctivity Ind

ex

0

2

4

6

8

10

12 Vehicle UDCA (30 mg/kg) UDCA (100 mg/kg) DSS (2.5 %) DSS + UDCA (30mg/kg) DSS + UDCA (100mg/kg)

***

***

*** *

**

n = 6-12

*UDCA compared to DSS

Time (days)

0 1 2 3 4 5 6

Disease activity in

dex

0

2

4

6

8

10

12 Control PBS DSS (2.5 %) DSS + 6MUDCA (30 mg/kg)

n = 3 - 9

Co

lon

leng

th (m

m)

0

20

40

60

80

n = 6 - 12, ANOVA + Tukey

* compared to DSS + Veh

*

Vehicle Vehicle+ UDCA(30 mg/kg)

+ UDCA(30 mg/kg)

+ DSS (2.5 %)

TNF-

pg

/ml

0

10

20

30

40n = 4, ANOVA + SNK

* compared to control

# compared to PIC #

UDCA - + - +PIC - - + +

***

***

B IL-8

pg

/ml

0

200

400

600

800

1000

1200

1400

UDCA - + - +PIC - - + +

***

***

n = 4, ANOVA + SNK

* compared to control

# compared to PIC#

IL-6

pg

/ml

0

100

200

300

400n = 4, ANOVA + SNK

* compared to control

UDCA - + - +PIC - - + +

***

**

IL-1

pg

/ml

0.0

0.5

1.0

1.5

2.0

2.5

**

UDCA - + - +PIC - - + +

***n = 4, ANOVA + SNK

* compared to control

# compared to PIC #

6MUDCA

C

D E

CVehicle

UDCA (30 mg/kg)

DSS (2.5%)+ Vehicle

DSS (2.5%) + UDCA(30 mg/kg)

UDCA ameliorates colitis in a mouse model of disease

SUMMARY & CONCLUSION

Time (days)

0 1 2 3 4 5 6

Disease A

ctivity Ind

ex

0

2

4

6

8

10

12Control LCA (30 mg/kg)DSS DSS + LCA (30 mg/kg)

******

n = 5, * DSS + LCAcompared to DSS

B

The UDCA metabolite, LCA prevents colitis in a mouse model of disease

Basal UDCA DSS DSS

[bile acid

] (M)

0

10

20

30

40UDCA CDCA LCA 3- 7-oxo

+ UDCA

******

***

**

Basal* vs respective basal bile acid n = 3, ANOVA + SNK, ** P 0.01, ** P 0.001

UDCA metabolites are elevated in a mouse model of disease

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.