tgr5 (gpbar1) and intestinal immunity
TRANSCRIPT
TGR5 (GPBAR1) a bile acid activated receptors
and intestinal inflammation
Stefano Fiorucci
University of Perugia Medical School
www.Stefanofiorucci.it
GP-BAR1 (TGR5, M-BAR1)
Energy
expenditure
Carbohydrate
Metabolism
TLCA-DCA
GP-BAR1
Bile Acids
Metabolism
Normal and
pathologic
growth and
differentiation
Immunity
0
1000
2000
3000
4000
*
GPBAR1-/-
GPBAR1+/+
(ng/m
l)
0.0
0.5
1.0
1.5
0
1
2
3
4
mR
NA
rela
tive e
xpressio
n
0.00
0.25
0.50
0.75
1.00
1.25
*
Zonulin-1 Ocludin E-cadherin Dextrane FITC
A
GPBAR1 +/+ GPBAR1 -/-
20x 20x
40x
20x
C GPBAR1-/- GPBAR1 +/+
D
10x 10x
20x
B
20x 20x
20x 20x
20x 20x
Cipriani S, Mencarelli A, Chini MG, Distrutti E, et al. (2011). PLoS ONE 6(10): e25637. doi:10.1371/journal.pone.0025637
0 1 2 3 40
1
2
3
4
GPBAR1-/-
GPBAR1+/+
*
Time (days)
Dia
rrhea s
core
0
20
40
60
80
100
GPBAR1 +/+
Naive DSS
GPBAR1 -/-
Naive DSS
MildAbsent Moderate Severe
Ma
cro
sco
pic
Da
ma
ge
(%
)
0
2500
5000
7500
10000
GPBAR1 +/+
Naive DSS
GPBAR1 -/-
Naive DSS
*
**
FIT
C-d
extr
an
e(n
g/m
l)
Naive DSS0.0
0.5
1.0
1.5
2.0
GPBAR1 +/+
*
GP
BA
R1
mR
NA
rela
tive
expre
ssio
n
DSS
GPBAR1 -/-
GPBAR1 -/- DSS
GPBAR1 +/+
GPBAR1 +/+ DSS
GPBAR1 +/+
Naive
A B C
D
F
20x 20x
20x
E
20x 10x
20x 20x
20x
Cipriani S, Mencarelli A, Chini MG, Distrutti E, et al. (2011) The Bile Acid Receptor GPBAR-1 (TGR5) Modulates Integrity of
Intestinal Barrier and Immune Response to Experimental Colitis. PLoS ONE 6(10): e25637. doi:10.1371/journal.pone.0025637
Naive TNBS DSS0.0
0.5
1.0
1.5
2.0
2.5
GPBAR1 +/+
* *
GP
BA
R1
mR
NA
rela
tive e
xp
ressio
nNormal Crohn's disease
0.0
0.5
1.0
1.5
2.0*
GP
BA
R1
mR
NA
rela
tive e
xp
ressio
n
20x
40x
Naive TNBS DSS
Crohn’s colitis
A C E
B D F
G
H I
L
Cipriani S, Mencarelli A, Chini MG, Distrutti E, et al. (2011) The Bile Acid Receptor GPBAR-1 (TGR5) Modulates Integrity of
Intestinal Barrier and Immune Response to Experimental Colitis. PLoS ONE 6(10): e25637. doi:10.1371/journal.pone.0025637
0
1
2
3
GPBAR1 +/+
Naive Ciprofloxacin TLCA
GPBAR1 -/-
*
*
Naive Ciprofloxacin TLCA
(10M) (10M)(10M) (10M)
Sple
en m
onocyte
scA
MP
[pm
ol/m
l]
0
25
50
75
100
*
**
*
*GPBAR1 +/+
Naive LPS Alone + Ciprofloxacin
GPBAR1 -/-
Naive LPS Alone + Ciprofloxacin
(10M) (10M)
TN
F
mR
NA
(rela
tive
expre
ssio
n)
NN
HN
F COOH
O
1 2
HO
NH
H
H
O
SO3
A
B
C
D
E
0
5
10
15
20
25
30
0 0.1 1 5 10 100
Ciprofloxacin (M)
[cA
MP
] i
(pg/m
g p
rote
in)
Naive
Cipro
floxa
cin
TLC
A
0
10
20
30
40
*
*
Cipriani S, Mencarelli A, Chini MG, Distrutti E, et al. (2011) The Bile Acid Receptor GPBAR-1 (TGR5) Modulates Integrity of
Intestinal Barrier and Immune Response to Experimental Colitis. PLoS ONE 6(10): e25637. doi:10.1371/journal.pone.0025637
0
1
2
3
4TNBS
*
**
-1 0 1 2 3 4 5
Naive TNBS TNBS +ciprofloxacin
Time (days)
Dia
rrhe
a s
core
Naive TNBS TNBS+Ciprofloxacin
10x 10x 10x
20x 20x 20x
0
1
2
3
4
*
**
Naive TNBS
Vehicle Ciprofloxacin
Mic
roscop
ic s
core
0
1
2
3
4
5
6 *
**
Ma
cro
sco
pic
sco
re
Naive TNBS CIPRO0
10
20
30
40*
**
IL1
rel.
exp
ressio
n
Naive TNBS CIPRO0.0
2.5
5.0
7.5
*
**
TN
Fa
rel.
exp
ressio
n
12
14
16
18
20
22
24
26
28
30
32
Naive TNBS TNBS + ciprofloxacin
TNBS
Ciprofloxacin or vehicle
**
*
-1 0 1 2 3 4 5
Time (Days)
We
igh
t (g
)
Cipriani S, Mencarelli A, Chini MG, Distrutti E, et al. (2011) The Bile Acid Receptor GPBAR-1 (TGR5) Modulates Integrity of
Intestinal Barrier and Immune Response to Experimental Colitis. PLoS ONE 6(10): e25637. doi:10.1371/journal.pone.0025637
Naive
LPS
TLCA 5
0M
LPS+T
LCA
Olene
alolic a
c 1
mM
LPS+
Olean
olic a
c0
1000
2000
3000
4000
5000*
****
RAW macrophages
TN
F
(pg/m
l)
0
10
20
30
0 5 10 100
Oleanolic acid (M)
[cA
MP
](p
g/m
g p
rote
in)
*
Cipriani S, Mencarelli A, Chini MG, Distrutti E, et al. (2011) The Bile Acid Receptor GPBAR-1 (TGR5) Modulates Integrity of
Intestinal Barrier and Immune Response to Experimental Colitis. PLoS ONE 6(10): e25637. doi:10.1371/journal.pone.0025637
0
1
2
3
*
** **
Naive TNBS
10 1 0.1 mg/kg
Oleanolic acid
Macro
scopic
inju
ry s
core
0 1 2 3 422
24
26
28
30
32
34
Naive TNBS alone TNBS + Oleanolic acid 10 mg
TNBS + Oleanolic acid 1 mg TNBS + Oleanolic acid 0.1 mg
*
*
Time (days)
We
igh
t (g
)
0 1 2 3 4 50
1
2
3
Time (days)
*D
iarr
he
a s
core
Cipriani S, Mencarelli A, Chini MG, Distrutti E, et al. (2011) The Bile Acid Receptor GPBAR-1 (TGR5) Modulates Integrity of
Intestinal Barrier and Immune Response to Experimental Colitis. PLoS ONE 6(10): e25637. doi:10.1371/journal.pone.0025637
0
10
20
30
40
Naive Naproxen Naive Naproxen
GPBAR1+/+
*
GPBAR1-/-
**
Inte
sti
nal
dam
ag
e(m
m o
f le
sio
n)
0
10
20
30
40
50
MP
O a
cti
vit
y(m
U/m
g p
rote
in)
*
*
Naive Naproxen Naive Naproxen
A
C
E
G
H
B
D
F
GP-BAR1-/- small intestine
GP-BAR1 gene deletion alters intestinal structure and function.
Cipriani S. et al. Activation of the bile acid receptor GPBAR1 protects against gastrointestinal injury caused by non-steroidal anti-
inflammatory drugs and aspirin in mice. Br J Pharmacol. 2013 Jan;168(1):225-37. doi: 10.1111/j.1476-5381.2012.02128.x.
Anti-inflammatory effects of oleanolic acid.
Cipriani S, Mencarelli A, Chini MG, Distrutti E, et al. (2011) The Bile Acid Receptor GPBAR-1 (TGR5) Modulates Integrity of
Intestinal Barrier and Immune Response to Experimental Colitis. PLoS ONE 6(10): e25637. doi:10.1371/journal.pone.0025637
20x
GPBAR1+/+ mouse naive GPBAR1 human
40x
20x
40x
A
B
C
D
GPBAR1
Monocytes Stomach0.0
0.5
1.0
1.5
mR
NA
rela
tive
exp
ressio
nCipriani S. et al. Activation of the bile acid receptor GPBAR1 protects against gastrointestinal injury caused by non-steroidal anti-
inflammatory drugs and aspirin in mice. Br J Pharmacol. 2013 Jan;168(1):225-37. doi: 10.1111/j.1476-5381.2012.02128.x.
0
10
20
30
40
50
60
GP-BAR1+/+
GP-BAR1-/-
Naive 10 mg/kg 30 mg/kg 50 mg/kg
ASA
**
**
*
**
**
Gastr
ic i
nu
ry s
co
re(m
m)
GPBAR1+/+ CTRL
GPBAR1-/- CTRL
GPBAR1+/+ ASA
GPBAR1-/- ASA
20x
20x
20x
20x
40x
H I
L M
Cipriani S. et al. Activation of the bile acid receptor GPBAR1 protects against gastrointestinal injury caused by non-steroidal anti-
inflammatory drugs and aspirin in mice. Br J Pharmacol. 2013 Jan;168(1):225-37. doi: 10.1111/j.1476-5381.2012.02128.x.
TNFα COX-1 COX-2
eNOS iNOS CSE CBS
0
1
2
3
4
*
GPBAR+/+
GPBAR-/-
*m
RN
A r
ela
tive e
xpre
ssio
n
0
1
2
3
mR
NA
rela
tive e
xp
ressio
n
0
1
2
3
4
5
6
*
**
mR
NA
rela
tive e
xp
ressio
n
0
1
2
3
4
5
6
mR
NA
re
lative e
xpre
ssio
n
*
0.00
0.25
0.50
0.75
1.00
mR
NA
re
lati
ve e
xp
ressio
n
*
*
0
1
2
3
Naive ASA Naive ASA
mR
NA
rela
tive e
xpre
ssio
n
*
*
0.0
0.5
1.0
1.5
*
*
Naive ASA Naive ASA
mR
NA
re
lative
exp
re
ssio
n
Cipriani S., et al. British Journal of Pharmacology 2012; 168: 225-237
Activation of the bile acid receptor GPBAR1 protects against gastrointestinal injury caused by non‐steroidal anti‐inflammatory drugs and aspirin in mice
MPO PGE2 TNFα
eNOS MUC1 MUC6
CSE CBS
0
10
20
30
40
Gastr
ic m
uco
sal
inju
ry s
co
re (
mm
lesio
n)
ASA 50 mg/kg
Betulinic acid - + - +
*
*
0
1000
2000
* * *
*
(pg/
mg
prot
ein)
0.00
0.25
0.50
0.75
1.00
1.25
1.50
mR
NA
rela
tive
expre
ssio
n
0
1
2
3
4
5
6
7*
*
mR
NA
rela
tive e
xpre
ssio
n
0
1
2
3
4*
Naive ASA ASA Naive ASA ASA + + Betulinic Betulinic
mR
NA
rela
tive e
xp
ressio
n
0
1
2
3
**
*
Naive ASA ASA Naive ASA ASA + + Betulinic Betulinic
0.00
0.25
0.50
0.75
1.00
1.25
**
Naive ASA ASA Naive ASA ASA
+ +
Betulinic Betulinic
0
1
2
3
4
5
*
0
10
20
30
40
50
MP
O(m
U/m
g p
ro
t) * *
Cipriani S., et al. British Journal of Pharmacology 2012; 168: 225-237
Betulinic acid a GPBAR1 agonist protects against gastrointestinal injury caused by non‐steroidal anti‐inflammatory drugs and aspirin in mice