Oxford Inflammatory Bowel Disease & Hepatology MasterClass

Gut and Liver Cross Talk

Professor David Adams

Gut

Liver

Transport via

portal circulation

Hepato-enteric immune system

Gut

Liver

Transport via

portal circulation

• Absorption of food

•Constant exposure to microbes

• Antigen presentation by

professional APC

Strong immune responses

• Metabolic processing of food

•Xenobiotic processing by hepatocytes

• Relatively less pathogen exposure

• Antigen presentation by non

professional APC – endothelium &

hepatocytes

Tolerance

Hepato-enteric immune system

Gut

Liver

Transport via

portal circulation

Hepato-enteric immune system

Co-ordinated immune response to shared

pathogens – a role for mucosal lymphocytes

Trichinosis – Trichinella Spiralis

Trichinella spiralis new born larvae (NBL) and

the hepato-enteric immune response

Liver tolerance to

new borne larvae

Effector CD4 cells

NBL

Trichinella spiralis new born larvae (NBL) and

the hepato-enteric immune response

Liver tolerance to

new borne larvae

Effector CD4 cells Effector CD4 cells

IL-10 deficient

Severe Hepatitis to

new borne larvae

NBL NBL

Trichinella spiralis new born larvae (NBL) and

the hepato-enteric immune response

Liver tolerance to

new borne larvae

Effector CD4 cells Effector CD4 cells No effector CD4 cells

IL-10 deficient

Severe Hepatitis to

new borne larvae

NBL

NBL NBL

Liver tolerance to

new borne larvae

Bliss,SK JI 2007

IL-10 deficient

Inflammatory bowel disease & liver disease

• Primary sclerosing cholangitis or autoimmune hepatitis develop in

– 3-10% of patients with IBD

– 70% of patients with PSC suffer from IBD – Broome et al Dis Col Rec 1995; Loftus Gut 2005

IBD PSC

Diseases

associated with

active bowel

disease

Pyoderma gangrenosum

Pyoderma gangrenosum

Erythema nodosum

Episcleritis

Liver disease runs a course that is independent

of bowel disease

•PSC can develop after colectomy for UC

•UC can develop after liver transplant for PSC

Alabraba et al Liver Tp 2008

Colectomy in the pre or peri-transplant period

protects against recurrence of PSC after liver

transplantation

Colectomy pre liver Tp

Colectomy post liver Tp

No Colectomy

IBD PSC

Do mucosal lymphocytes cause liver disease ?

Hypothesis:

Gut activated lymphocytes in IBD enter the liver and contribute to

liver inflammation in PSC.

Small bowel

Peyer’s patch

DC

migration

CCL25 Madcam-1

Mesenteric LN

Mucosal venule

Adaptive immune responses in the gut

Adams & Eksteen,

Nature Rev Immunol

2006

Small bowel

Peyer’s patch

DC

migration

CCL25 Madcam-1

Mesenteric LN

Mucosal venule

Adaptive immune responses in the gut

Adams & Eksteen,

Nature Rev Immunol

2006

Small bowel

Peyer’s patch

DC

migration

CCR9+ a4b7+

CCL25 MAdCAM-1

Mesenteric LN

Mucosal venule

Adaptive immune responses in the gut

Adams & Eksteen,

Nature Rev Immunol

2006

Imprinting of

a4b7 and

CCR9 confers

gut homing

Small bowel

Peyer’s patch

DC

migration

CCR9+ a4b7+

Mesenteric LN

Mucosal venule

Adaptive immune responses in the gut

Adams & Eksteen,

Nature Rev Immunol

2006

CCL25 MAdCAM-1

CCL25

Small bowel

Peyer’s patch

DC

migration

CCR9+ a4b7+

CCL25 Madcam-1

Mesenteric LN

Mucosal venule

Adaptive immune responses in the gut

Adams & Eksteen,

Nature Rev Immunol

2006

Small bowel

Peyer’s patch

DC

migration

CCR9+ a4b7+

CCL25 Madcam-1

Mesenteric LN

Mucosal venule

Adaptive immune responses in the gut

Adams & Eksteen,

Nature Rev Immunol

2006

CCL25

CCL25 MAdCAM-1

Gut dendritic cells induce expression of a4b7 on T cells

Stagg et al Eur J Immunol 2003

Gut dendritic cells imprint lymphocytes with the

unique mucosal homing phenotype

Selective generation of gut tropic T cells in gut-associated

lymphoid tissue

Johansson-Lindbom,B J Exp. Med 2003

Peyer’s Patch DCs imprint gut homing on lymphocytes

whereas DCs from spleen or PLN cannot

Mora et al Nature 2003

Retinoic acid imprints gut-homing specificity on T cells

Iwata M, Hirakiyama A, Eshima Y, Kagechika H, Kato C, Song SY.

Immunity Oct 2004

•Retinoic acid, enhances expression of a4b7 and CCR9 on T cells

and imprints them with gut tropism.

•Vitamin A deficiency causes a reduction in a4b7 memory T cells

in lymphoid organs and intestinal lamina propria.

Novel role for retinoic acid in imprinting

gut-homing specificity on T cells

Retinoic acid imprints gut-homing specificity on T cells Iwata M et al Immunity Oct 2004

Retinal Retinol Retinoic acid

ADH Retinal

dehydrogenase

Retinoic acid imprints gut-homing specificity on T cells Iwata M et al Immunity Oct 2004

Retinal Retinol Retinoic acid

ADH Retinal

dehydrogenase

Selectively expressed

in gut DCs

Retinoic acid imprints gut-homing specificity on T cells Iwata M et al Immunity Oct 2004

Retinal Retinol Retinoic acid

ADH Retinal

dehydrogenase

RAR

Retinoic acid

receptors

Selectively expressed

in gut DCs

Retinoic acid imprints gut-homing specificity on T cells Iwata M et al Immunity Oct 2004

Retinal Retinol Retinoic acid

RA-responsive

enhancer sequence

CCR9

ADH Retinal

dehydrogenase

RAR

Retinoic acid

receptors

3p21.3

b7 Chromosome 15

Selectively expressed

in gut DCs

MLN DC 71%

Unstimulated 8.8%

Isotype

matched

control mab

CC

R9

Activation of CD3+ PBL at 7 days

CD3/CD28

100nM ATRA

66.4%

(52.6)

Generation of Gut-Homing IgA-Secreting B Cells by

Intestinal Dendritic Cells

Mora, Eksteen et al Science 2006

DCs from human mesenteric LN imprint

CCR9 on responding lymphocytes

Constitutively expressed in the liver

Supports lymphocyte recruitment McNab Gastro 1996; Lalor J Immunol 2002; Bonder et

al Immunity 2005

Increased in mesenteric vessels in IBD Grant 2002; Salmi 1998

Vascular adhesion protein 1 promotes

lymphocyte recruitment to the liver

Contact Arrest

Extravasation

Sinusoidal endothelium

VELOCITY VAP-1

ICAM-1 VAP-1

VAP-1 in normal human liver

VCAM-1 MAdCAM-1

α4β1

α4β7

CCR9

ICAM-1

αLβ2

Gut Endothelial cell

Gut homing lymphocytes Liver infiltrating lymphocytes

VAP-1R

VAP-1

VAP-1R

CXCR3

CXCL10 CCL25

Hepatic Endothelial cell

MLN

Retinoic acid

Gut DC

MAdCAM-1 (VAP-1)

α4β7

CCR9

VAP-1

MAdCAM-1 (VAP-1)

α4β7

CCR9

MLN

MADCAM-1

PORTAL LN

a4b7+ T cells in the

PSC liver

Grant,A.J. et al. 2001 Hepatology.

CD3

α4β7

MAdCAM-1 in PSC

MAdCAM-1

MAdCAM-1

CCL25

α4β7

CCR9

MLN MADCAM-1 and CCL25 upregulated on

endothelium in PSC and autoimmune hepatitis Eksteen et al J Exp Med 2004

CCL25

Merged CCL25

CD31

In PSC 20% of liver lymphocytes express CCR9

+

0 20 40 60 80 100 Mean % CCR9+ T-cells

CD LPL n=3

CD PBL n=3

NL LIL n=6

N PBL n=6

PBC LIL n=6

PSC PBL n=8

PSC LIL n=8

a4b7

CC

R9

PSC LIL 17%

In PSC 20% of liver lymphocytes express CCR9

+

0 20 40 60 80 100 Mean % CCR9+ T-cells

CD LPL n=3

CD PBL n=3

NL LIL n=6

N PBL n=6

PBC LIL n=6

PSC PBL n=8

PSC LIL n=8

a4b7

CC

R9

PSC LIL 17%

In PSC 20% of liver lymphocytes express CCR9

+

0 20 40 60 80 100 Mean % CCR9+ T-cells

CD LPL n=3

CD PBL n=3

NL LIL n=6

N PBL n=6

PBC LIL n=6

PSC PBL n=8

PSC LIL n=8

PBL

IFNγ

PHA 0hrs

PHA 0hrs

PHA 4hrs

PHA 4hrs

1%

13%

78%

2%

These are primed T cells

+CCL25 CCL25 + PTX Control

% c

on

tro

l m

igra

tio

n

80

90

100

110

120

* * p<0.01

*

Miles et al Am J Phys 2008

Transfection of human hepatic sinusoidal endothelial

cells (HSEC) with MAdCAM-1 allows them to support

migration of T cells isolated from PSC liver in the

presence of CCL25 MAdCAM-1

140

What induces the expression of

MADCAM-1 and CCL25 in the

liver?

MAdCAM-1 staining in inflamed PSC liver A. small B. large portal vessels

VAP-1

(SSAO)

RGD motif

Endothelium

Lymphocyte

Amine

Aldehyde

NH3

H2O2

?

Activation

VAP-1:Ligand

interaction

VAP-1 is an adhesion molecule on liver endothelium

It is also an amine oxidase

Amines from

food and wine

and bacteria

Salmi Science 1991; Smith JEM 1998 Jalkanen and Salmi EMBOJ 2001

RGD motif

Endothelium

Lymphocyte

Amine

Aldehyde

NH3

H2O2

?

NFkB Activation

VAP-1:Ligand

interaction

How does enzyme activity affect adhesion?

TRANSCRIPTION OF

CHEMOKINES AND ADHESION

MOLECULES

Lalor et al Hepatology 2007; Liaskou et al Hepatology 2011

MAdCAM-1 is induced on liver endothelium by methylamine & TNFa A

dh

ere

nt

ce

lls

P=0.01 P=0.018

0 10

20 30 40 50 60

Control TNFa TNFa+ TNFa+Meth

Methylamine +anti MAdCAM-1

Liaskou et al Hepatology 2011

Feeding a VAP-1 enzyme substrate methyl amine to mice that over-

express VAP-1 results in increased MADCAM-1 expression in the gut

Liaskou et al Hepatology 2011

Induction of MADCAM-1

Natural amines from gut via portal vein

activate VAP-1

+

Pro-inflammatory cytokines

NFkB activation

MAdCAM-1 induction

VCAM-1 MAdCAM-1

α4β1

α4β7

CCR9

VCAM-1

α4β1

α4β7

MAdCAM-1

Endothelial cell

Gut homing lymphocytes PSC Liver infiltrating lymphocytes

VAP-1R

VAP-1

VAP-1R

CCR9

CCL25 CCL25

Where are gut-homing T cells in PSC

imprinted?

In the liver or the gut?

Neo lymphogenesis is seen in portal-associated

lymphoid follicles in PSC

CCL21 on HEV

CCL19 on

lymphatics and

stromal cells

Grant et al Am J Path 2002

Low levels of functional CCR9 induced by liver DCs in

the absence of ATRA

Low levels of functional CCR9 induced by liver DCs in

the absence of ATRA

Eksteen et al Gastro 2009

VCAM-1 MAdCAM-1

α4β1 α4β7

CCR9

VCAM-1

α4β1

α4β7

MAdCAM-1

Endothelial cell

Gut homing lymphocytes PSC Liver infiltrating lymphocytes

VAP-1R

VAP-1

VAP-1R

CCR9

CCL25 CCL25

DCs from human liver CANNOT imprint high levels of

functional CCR9 on responding lymphocytes

VCAM-1 MAdCAM-1

α4β1 α4β7

CCR9

VCAM-1

α4β1

α4β7

MAdCAM-1

Endothelial cell

Gut homing lymphocytes PSC Liver infiltrating lymphocytes

VAP-1R

VAP-1

VAP-1R

CCR9

CCL25 CCL25

CCR9+ T cells in the liver are likely to

be mucosal T cells imprinted by gut DC

Under some circumstance T cell activation in the liver

can induce gut homing

Connecting liver and gut: murine liver sinusoidal endothelium

induces gut tropism of CD4+ T cells via retinoic acid.

Neumann K, Kruse N, Szilagyi B, Erben U, Rudolph C, Flach A, Zeitz

M, Hamann A, Klugewitz K. Hepatology. 2012 Jun;55(6):1976-84.

Hepatology. 2012 Jun;55(6):1976-84.

IBD

Naïve T cell

MLN + PPs

a4b7

Naïve T cell

Effector T cell

VAP-1r

MLN + PPs

CCR-9 CXCR3

IBD

a4b7

Liver endothelium Gut endothelium

VAP-1, MAdCAM-1 MAdCAM VAP-1

CCL25, CXCR3

Naïve T cell

VAP-1r

MLN + PPs

CCR-9 CXCR3

IBD

a4b7

Naïve T cell

Memory T cell

VAP-1r

MLN + PPs

CCR-9

Liver endothelium Gut endothelium

VAP-1, MAdCAM-1 MAdCAM

CCL25, CCL28

GUT

a4b7

Liver endothelium Gut endothelium

VAP-1, MAdCAM-1 MAdCAM

NO antigen

NO retention

release to lymphatics

RECIRCULATION

Naïve T cell

Memory T cell

VAP-1r

MLN + PPs

a4b7 VAP-1r

Naïve T cell

Memory T cell

MLN + PPs

+antigen

activation

retention

INFLAMMATION

a4b7 VAP-1r

CCR-9

a4b7 VAP-1r

CCR9

GUT

VAP-1, MAdCAM-1 MAdCAM

Naïve T cell

Memory T cell

MLN + PPs

GUT

Liver endothelium Gut endothelium

MAdCAM-1 CCL25 MAdCAM, VAP-1

a4b7 VAP-1r

MAdCAM

CCL25

CCL28

CCR9

+antigen

activation

retention

INFLAMMATION

a4b7 VAP-1r

CCR9 CXCR3

Naïve T cell

Memory T cell

MLN + PPs

GUT

Liver endothelium Gut endothelium

MAdCAM-1 CCL25 MAdCAM, VAP-1

a4b7 VAP-1r

MAdCAM

CCL25

CCL28

CCR9

+antigen

activation

retention

INFLAMMATION

a4b7 VAP-1r

CCR9 CXCR3

Inhibitors of CCR9 and a4b7

currently in clinical trials for IBD

Will they work in PSC?

CREDITS

Collaborators

Sirpa Jalkanen and Marko Salmi (Turku)

Ron Gladue and Nick Pullen (Pfizer)

Mike Briskin (Merrimack Pharma)

Uli von Andrian and Rodrigo Mora (Harvard)

THE UNIVERSITY OF BIRMI NGHAMBirmingham

Liver Labs

Bertus Eksteen

Allister Grant

Trish Lalor

Lia Liaskou

Palak Trivedi

Stuart Curbishley

Alice Miles

Chris Weston

Dept Pathology

Stefan Hubscher