Gut Immunity - icds.si · Gut Immunity Shomron Ben-Horin Laboratory of Gastro-Immunology Gastroenterology Department Sheba Medical Center, Tel-Aviv University, Israel. Disclosures

Gut Immunity

Shomron Ben-Horin

Laboratory of Gastro-Immunology

Gastroenterology Department

Sheba Medical Center, Tel-Aviv University, Israel

Disclosures

Dr. Shomron Ben-Horin received consultancy fees from:

Abbot Laboratories, Schering-Plough, Optimer Inc.

Immunology made easy

Thymocytes Naïve

CD4+ T cellTh17/Th

IL-17

IL-17F

IL-6

TNF-a

IL-23

IL-23R

IFN

-g

IL-4

IFN-g

IL-10

TGF-b1

IL-4

IL-5

IL-13STAT6

GATA3

Treg

Th2

IL-4R

CD25

FoxP3

Th1

IL-12R

STAT1

STAT4

T-bet

TGF-b1

IL-6

Human host response after exposure

to a typical immunology slide

Let’s start with a brief review of :

Gut immune system organization

Gut immune system functions

Traditional view -1

The epithelial functions consist of physical barrier and absorption apparatus

Traditional view 1: epithelium is only a barrier…

Traditional view -2

IBD is caused by exaggerated gut immune response (but CDAD is not….)

Traditional view 2: Crohn’s disease is an immune hyper-activity disorder…

Immune in-adequacy Immune hyper-reactivity

Traditional view -3

Immune memory is antigen specific

Traditional view 3: Immune memory is only antigen-specific….

Intestinal Mucosa Defense- Organization

Non-Immune

Immune

Two types of mechanisms to control pathogens,

normal flora (1014) & dietary molecules

Intestinal Mucosa Defense: Non-immune

Response type - Immune reaction:

Innate response

Adaptive response

Topographic - Immune compartmentalization:

Intestinal Mucosa Defense: Immune

Topographic Organization of GALT

Mesenteric lymph nodes

Peyer’s Patches

Cryptopatches

Solitary lymphoid follicles

Solitary immune cells:

Intraepithelial lymphocytes (IELs)

Lamina propria lymphocytes (LPLs)

Atkins D, Nature Rev Immunol 2009

Peyer’s patchDome-like protruding into lumen

Sub-epithelial dome (SED) -

3-4 Follicles (B cell germinal center), inter-follicular region

(T-cells), high endothelial venules (HEV) & lymphatics

Villous epithelium & Microfold cells (M cells)

M cells: No villi, thin glycocalyx, basally folded around immune cells. Role: Uptake of Antigens &Transcytosis

Neutra MR, Nature Immunol 2001

Peyer’s patchDome-like protruding into lumen

Sub-epithelial dome (SED) -

3-4 Follicles (B cell germinal center), inter-follicular region

(T-cells), high endothelial venules (HEV) & lymphatics

Villous epithelium & Microfold cells (M cells)

M cells: No villi, thin glycocalyx, basally folded around immune cells. Role: Uptake of Antigens &Transcytosis

M cell

Jang MH, PNAS 2004

Other inductive sites

Cryptopatches

Clusters of hematopoeitic progenitors and dendritic cells

Role: Extra-thymic development of T-cells

Solitary follicles

Clusters of lymphocytes - B cells and dendritic cells

Antigen sampling and presentation >> IgA production

Topographic Organization of GALT -

Effectors

Solitary

follicle

Peyer’s patch

Cryptopatch

Paneth Cell

Isolated Lymphocytes

Intra-epithelial lymphocytes (IELs) – CD8+ and gd T-cells

Lamina propria lymphocytes (LPLs) – CD4+ T cells & B-cells

Primed antigen-specific effector cells:

Excrete pro-inflammatory cytokines

Exert T-cell cytocytoxicity

Secrete Immunoglobulines (IgA)

Atkins D, Nature Rev Immunol 2009

Suzuki K, Trends Immunol 2008

Secretory IgA (& pentameric IgM) produced by LP

B-cells and actively secreted to gut lumen by epithelium

Topographic Organization of GALT

Mesenteric lymph nodes

Peyer’s Patches Inductive

Cryptopatches sites

Solitary lymphoid follicles

Solitary immune cells:

Intraepithelial lymphocytes (IELs) Effector

Lamina propria lymphocytes (LPLs) sites

Atkins D, Nature Rev Immunol 2009

Intestinal effector T & B cells – Education/migration

1. Naïve T & B cells presented with antigen by

macrophages/DCs in Peyer’s patches or mesenteric lymph

nodes >> effector cells

2. Exit through efferent lymph vessels into circulation/spleen

So how do they return to intestinal wall ?

Migration of Cells into Tissues

Selectin ligands,L-SelectinCCR 9

E, P Selectins

Mucosa

ACTIVATION ARRESTROLLING

Integrins

a4b7Adhesion MoleculeMADCAM-1

TRANSMIGRATION

Cytokines &

chemokines

(TECK - CCR9

ligand)

Oral immunization with S.typhy Ty21a induce antigen-responsive cells expressing a4b7 integrin, whereas

parenteral vaccination does not

Traditional view 1: Immune memory is only antigen-specific….

Kantele A, J Immunol 1999

0

20

40

60

80

100

Pa

tie

nts

in

re

mis

sio

n

(%)

L-sel

ectin

a4b7

inte

grin

Oral immunization

Parenteral

Un-vaccinated

P=<0.001 P=0.01

% A

ntigen-r

esponsiv

e c

ells

Intestinal dendritic cells in Peyer patches & mesenteric LN prime memory T-cells to express gut-homing molecules:

a4b7 integrin and CCR9 chemokine receptor

Traditional view 1: Immune memory is only antigen-specific….

Mora JR, Nature 2003

Campbell DJ, J Exp Med 2002

Johansson-Lindbom B, J Exp Med 2003

CD4+ cells primed by dendritic cell in the mesenteric lymph nodes preferentially migrate back to the GALT

Traditional view 1: Immune memory is only antigen-specific….

Iwata M, Immunity 2004

CD4+ cells primed by MLN DC (labeled with green CFSE)

or by PLN DC (red – TRITC labeled)

After injection into mice

>>

T cells primed by MLN DC(Green labeled) are

Much more able to migrate back to Peyer Patch

Media

Gliadin-TTG

TT

0.9%

5.1%

2.6%

3.8%

7.1%

0.8%

b7integrin

CFSE

Ben-Horin S et al, J Leuk biol 2006

Gliadin-TTG specific CD4+ memory T-cells, but not Tetanus

specific cells, preferentially express the gut-homing b7 integrin

Traditional view 1: Immune memory is only antigen-specific….

Media

Gliadin-TTG

TT

0.9%

5.1%

2.6%

3.8%

7.1%

0.8%

b7integrin

CFSE

Ben-Horin S et al, J Leuk biol 2006

Gliadin-TTG specific CD4+ memory T-cells, but not Tetanus

specific cells, preferentially express the gut-homing b7 integrin

NEW view 1: Immune memory is antigen-specific….AND tissue-specific

Feagan B, N Engl J Med 2005

Anti a4b7 integrin mAb (Vedolizumab) induces remission

in moderately active ulcerative colitis

Traditional view 1: Immune memory is only antigen-specific….

0

10

20

30

40

Pa

tie

nts

in

rem

iss

ion

(%

)

Vedol

izum

ab 2

mg/

kg

Vedol

izum

ab 0

.5m

g/kg

Place

bo

P=0.02P=0.03

Intestinal epithelial cells (m-IC) induce expression of gut homing

phenotype by T-cells in the absence of intestinal dendritic cells

Edele F, J Immunol 2008

Traditional view 2: epithelium is only a barrier…

CD8+ cells only

CD8+ cells &

bone marrow DC

CD8+ cells &

bone marrow DC &

gut epithelial cells

CD8+ cells &

bone marrow DC &

gut epithelial cells (transwell)

Topographic Organization of GALT

Mesenteric lymph nodes

Peyer’s Patches Inductive

Cryptopatches sites

Solitary lymphoid follicles

Solitary immune cells:

Intraepithelial lymphocytes (IELs) Effector

Lamina propria lymphocytes (LPLs) sites

Epithelium ??

Intestinal Epithelium expresses

bacterial sensing molecules

Traditional view 2: epithelium is only a barrier…

Giraradin SE, Science 2003

Intestinal Epithelium expresses

bacterial sensing molecules

Traditional view 2: epithelium is only a barrier…

NOD1 – Recognition of Gram-

bacterial wall tri-peptide

NOD2- Recognition of Gram-/+

bacterial wall MDP

>> Activation of MAPK-NFkB pathways

& Secretion of chemokines/peptides

Giraradin SE, Science 2003

Peptidoglycan (G+)LipoproteinLipoarabinomannan(Mycobacterial)LPS (Leotosopia)LPS (Porphyromonas)GPI (Trypanosoma cruzi)Zymosan Yeast)

LPS (G-)Lipoteichoic acids (G+) RSV F Protein

dsRNA Flagellin Unmethylated CpG DNA

TLR9TLR5TLR3TLR4CD14TLR1TLR2TLR6

MD-2

Toll-Like Receptors: Recognition of Pathogen

associated molecular patterns PAMP)

Artis D, Nature Rev Immunol 2008

Su

rviv

al

(%)

Deficient TLR signaling increases

mortality after DSS colitis

Depletion of intestinal flora

increases mortality after DSS colitis,

and LPS restores protection

Rakoff-Nahoum S, Cell 2004

Day 0 3 6 9 12 15 18 21

100

80

60

40

20

0

A

WT

MyD88-/-

TLR4-/-

TLR2-/-

B

Commensal triggering of TLR maintains epithelial

homeostasis and protects against DSS colitis

Traditional view 2: epithelium is only a barrier…

Selective NFkB ablation in intestinal epithelium of mice by knocking out NEMO (IKK-g) leads to innate and later adaptive

immune cell inflitrate and intestinal inflammation

Traditional view 2: epithelium is only a barrier…

Nenci A, Nature 2007

Murine models validity for human studies

Arch Gynecol - Links

The effect of intraperitoneal colchicine on the formation of peritoneal adhesions in the rat

Shapiro I, Granat M, Sharf M.

A double blind study was carried out to determine the effect of

colchicine on the formation of intraperitoneal adhesions in the rat. Adhesions were produced by a standard method in 92 rats.

The animals were than divided at random into two groups. One group (47 rats) was treated with intraperitoneal colchicine, whilst the other, the control group (45 rats) was treated with intraperitoneal normal saline in the same volume, frequency and duration.

Two kinds of adhesions were found. 1. "Surface adhesion" - consisting of two serosal surfaces attached together; their area was significantly smaller (p less than 0.00003), and their number was lower (p less than 0.03) in the colchicine group than in the control group. 2. "Filamentous adhesions" - thin, elastic cords connecting omentum or pelvic fat bodies to other viscera. We concluded that post-operative treatment with colchicine reduces the formation of intraperitoneal adhesions in the rat

Murine models validity for human studies

Arch Gynecol - Links

The effect of intraperitoneal colchicine on the formation of peritoneal adhesions in the rat

Shapiro I, Granat M, Sharf M.

A double blind study was carried out to determine the effect of

colchicine on the formation of intraperitoneal adhesions in the rat. Adhesions were produced by a standard method in 92 rats.

The animals were than divided at random into two groups. One group (47 rats) was treated with intraperitoneal colchicine, whilst the other, the control group (45 rats) was treated with intraperitoneal normal saline in the same volume, frequency and duration.

Two kinds of adhesions were found. 1. "Surface adhesion" - consisting of two serosal surfaces attached together; their area was significantly smaller (p less than 0.00003), and their number was lower (p less than 0.03) in the colchicine group than in the control group. 2. "Filamentous adhesions" - thin, elastic cords connecting omentum or pelvic fat bodies to other viscera. We concluded that post-operative treatment with colchicine reduces the formation of intraperitoneal adhesions in the rat

Paneth cell

Specialized epithelial cells at base of crypts

Respond to certain patterns of bacteria

Secrete anti-bacterial peptides (Defensins)

Traditional view 2: epithelium is only a barrier…

NOD2 Knockout mice have defective expression of

Defensins after Listeria infection

Does the Nod2 defect induce loss of function and

reduces innate immunity exposure to bacterial

infections mainly in the TI region?

Kobayashi KS, Science 2005

Rela

tive e

xp

ressio

n

1.00E+05

1.00E+04

1.00E+03

1.00E+02

1.00E+01

infection- +- +

WT Nod2-/-

1.00E+05

1.00E+04

1.00E+03

1.00E+02

1.00E+01

infection- +- +

WT Nod2-/-

60

50

40

30

20

10

0

infection - +- +WT Nod2-/-

Rela

tive e

xp

ressio

n

Rela

tive e

xp

ressio

n

Defcr5Defcr-rs10Defcr4

Traditional view 2: epithelium is only a barrier…

Paneth cell

NOD2 mutations found in 15% Crohn’s patients

and associated with diminished mucosal

a-defensin expression

>> reduced control of luminal bacteria ??

% o

f N

on

-dis

ease C

on

tro

l

HD5 Peptidegg/mg extract

100

80

60

40

20

0CD CD

(NOD2-wt) (NOD2-mut)Wehkamp J, PNAS 2005

Traditional view 2: epithelium is only a barrier…

WT bone marrow transfer does not restore function >> Epithelial dependency

NOD2- mice develop H. hepaticus granulomatous ileitis in

Peyer patches, reversed in transgenic human defensin mice

Biswas A, PNAS 2010

Traditional view 2: epithelium is only a barrier…

The a-defensins impact the microbial composition of the gut

Salzmann NH, Nature Immunol 2010

Traditional view 2: epithelium is only a barrier…

Defensin Knock-outs Transgenic defensin mice

Pre-incubation of intestinal epithelium with a-defensins ameliorates C.difficile Toxin B injury

Traditional view 2: epithelium is only a barrier…

Giesmann T, Gastroenterology 2008

Pre-incubation of intestinal epithelium with a-defensins ameliorates C.difficile Toxin B injury

NEW view 2: epithelium is a barrier…but also an immune organ

Giesmann T, Gastroenterology 2008

NEW view 2: epithelium is a barrier…but also an immune organ

Intestinal enterocytes induce expression of FoxP3 in activated

CD4+ cells and expansion of Treg population

Dotan I, Am J Gastrointest Physiol, 2008

Intestinal enterocytes up-regulate MICA expression and IL-15

secretion leading to Intra-epthelial CD8+ T-cell activation and

epithelial damage after exposure to gliadin in celiac

Kagnoff MF, J Clin Invest 2007

Ben-Horin S et al, Submitted

CFSE

CD

45R

O

Media GTG GTG-Trypsin

3%

3% 5%3%

4%3%

Index patient

4%

3%

19%

3%

17%

3%CD

45R

O

Celiac patient

In a bone-marrow transplanted celiac :No immune memory to celiac antigens

NEW view 2: epithelium is a barrier…but also an immune organ

Ben-Horin S et al, Submitted

In a bone-marrow transplanted celiac :Intestinal epithelial-lymphocyte chimerism

NEW view 2: epithelium is a barrier…but also an immune organ

11

10

9

8

7

6

5

4

3

2

1

0

-1

1 10 100 1000

wt/wt

702Trp/702Trp

702Trp/1007fs

908Arg/1007fs

1007fs/1007fs

MDPmg/L

IL8 n

g/m

l

Mutated NOD2 Monocytes Mount Defective Immune

Responses: Innate system dysfunction in Crohn’s disease?

Van heel D, Lancet 2005

Traditional view 3: Crohn’s disease is an immune hyper-activity disorder…

PBMCs’ response to MDP and peptidoglycans is reduced in Crohn’s patients homozygous for the NOD2 mutation (Nod2fs) compared to controls (CTR) & Crohn’s patients

without defects in Nod2 (Crohn)

Netea MG, J Biol Chem 2005

Traditional view 3: Crohn’s disease is an immune hyper-activity disorder…

Seidelin A, PLoS One 2009

Diminished response to MDP is also present in monocytes

from Crohn’s patients without NOD2/CARD15 mutations

Traditional view 3: Crohn’s disease is an immune hyper-activity disorder…

NOD2 activating mutations cause constitutive NFkB

activation and early-onset sarcoidosis (Blau syn)

Traditional view 3: Crohn’s disease is an immune hyper-activity disorder…

Okafuji I, Arthritis Rheum 2009

Van Beelen AJ, Immunity 2007

TLR2 & NOD2 signaling cause dendritic cells to induce IL-17

expression by memory T-cells

Traditional view 3: Crohn’s disease is an immune hyper-activity disorder…

Yen D, J Clin Invest 2006

Th17 memory T-cells are induced by IL-23 and mediate

intestinal inflammation in IL-10 KO mice model

Traditional view 3: Crohn’s disease is an immune hyper-activity disorder…

Secretion of IL17 by lamina propria mononuclear cells (LPMCs) is increased in patients with Crohn’s disease (CD) & ulcerative colitis (UC) compared to non-IBD patients patients

and healthy controls (HC)

Rovedatti L , Gut 2009

Cadwell K, Nature 2008

Defective Autophagy in ATG16L1 mutated mice & Crohn’s

patients results in disordered paneth cells and reduced

lysozyme secretion

Traditional view 3: Crohn’s disease is an immune hyper-activity disorder…

Wild type ATG knock-out

Crohn’s

without ATG16L1

mutation

Crohn’s

with ATG16L1

mutation

Kuballa P, PLoS One 2009

siRNA silencing of ATG16L1 results in reduced Salmonella

Typhimurium internalization into autophagosome

Traditional view 3: Crohn’s disease is an immune hyper-activity disorder…

Crohn’s patients in remission have decreased recruitment of PMN & reduced clearance of E.coli

injected into forearm

Smith AM, J Exp Med 2009

Traditional view 3: Crohn’s disease is an immune hyper-activity disorder…

Deficient Innate Immunity in Crohn’s disease ?

mucosa

tissue damageDeficient initial innate immunity: aggressive chronic adaptive response

GM-CSF Confers Therapeutic Advantage in

Crohn’s Disease

Day Day Day Follow-up15 29 57 day 30

60

50

40

30

20

10

0

Perc

en

tag

e o

f P

ati

en

ts

Primary End Point

(70-p0int decrease in CDAI)

Secondary End Point

(100-p0int decrease in CDAI)

Remission

(CDAI score 150 points)

P=0.08P=0.006 P=0.28

P=0.03

P=0.02

P=0.002

P=0.01

P=0.02

P=0.73

P=0.01

P=0.02

P=0.01

Sargramostim

Placebo

Day Day Day Follow-up15 29 57 day 30

Day Day Day Follow-up15 29 57 day 30

Korzenik JR, N Engl J Med 2005

NEW view 3: Crohn’s disease is an (innate) immune deficiency disorder…

TcdA & TcdB trigger IL-1b secretion by NLRP intra-cellular inflammasome, and disease is

reversed by IL-1b blockade

Ng J, Gastroenterology 2010

Traditional view 3: Crohn’s is immune hyper-activity….But CDAD is not….. ?

Transforming normal colon to inflammation

Immune in-adequacy Immune hyper-reactivity

Thank you for your attention